Welcome to IISER-TVM'S Lab Notebook Wiki! This digital notebook serves as a comprehensive record of experiments, observations, and research conducted in our laboratory. Whether you're a member of our team or an external collaborator, this wiki is designed to promote transparency and document our scientific journey. Feel free to explore this guide, we hope it will help you understand the significance of documentation and how to effectively utilize your lab notebook in your research endeavors.

Saturday - 8/7/2023

Making TE buffer for resuspension of IDT orders.

Required final concentrations:
EDTA 0.1mM
Tris HCl- 10mM

#This EDTA concentration is for oligo storage.
First step, making 100 ml of 1M tris HCl and 0.5M EDTA
1M Tris HCl
  • Weigh out 12.11 g Tris(Tris buffer) and add to a 100 mL beaker
  • Measure out 80 mL of distilled water and add to the beaker
  • Add a magnetic flea and place it on a magnetic stirring plate to mix the solution.
  • Add a pH meter to the solution to observe the pH
  • Slowly add concentrated hydrochloric acid (HCl) solution and make up to pH 8.
0.5M EDTA
  • Weigh out 18.61 g EDTA disodium salt, dihydrate and add to a 100 mL beaker.
  • Measure out 80 mL of distilled water and add to the beaker.
  • Add a magnetic bead and place it on a magnetic stirring plate to mix the solution. The EDTA salt will not go into solution until the pH reaches 8.0.
  • Add a pH meter to the solution to observe the pH
  • To dissolve the salt, add sodium hydroxide (NaOH) pellets to the solution. Add a few pellets at a time and wait until the pellets have fully dissolved before adding more. It may take around 2 g (way more) of NaOH pellets before the pH is at 8.0.
  • once close to 8 make a solution using pellets of naoh and slowly add the solution dropwise.
To make the TE buffer
  • Measure out 1 mL 1M Tris-Cl (pH 8.0) and add to a 100 mL beaker
  • Measure out 0.02 mL 0.5M EDTA (pH 8.0) and add to the beaker.
  • Top up the solution to 100 mL by adding distilled water.
  • Place the lid on the bottle and invert a few times to mix.
  • To sterilise, autoclave the solution on a liquid cycle (20 min at 15 psi).
  • Autoclaved everything and kept it at room temperature
    • (to do- label everything with new labels and store)

Tuesday - 7/8/2023

  • Prepared IDT order google sheet: IDT order and resuspension details


 
  • We got advice from Nishana ma'am and Swathi ma'am to use TE buffer to resuspend the oligos obtained from IDT
  • TE buffer prep:
    • required final concentrations-
EDTA 0.1mM
Tris HCl- 10mM
###This EDTA concentration is for oligo storage
FIRST step, making 100 ml of 1M tris HCl and 0.5M EDTA
 
1M Tris HCl
Weigh out 12.11 g Tris(Tris buffer) and add to a 100 mL beaker
Measure out 80 mL of distilled water and add to the beaker
Add a magnetic flea and place on a magnetic stirring plate to mix the solution.
Add a pH meter into the solution to observe the pH
Slowly add concentrated hydrochloric acid (HCl) solution and make upto pH 8.
 
 
0.5M EDTA
Weigh out 18.61 g EDTA disodium salt, dihydrate and add to a 100 mL beaker.
Measure out 80 mL distilled water and add to the beaker.
Add a magnetic bead and place on a magnetic stirring plate to mix the solution. The EDTA salt will not go into solution until the pH reaches 8.0.
Add a pH meter into the solution to observe the pH.
To dissolve the salt, add sodium hydroxide (NaOH) pellets to the solution. Add a few pellets at a time and wait until the pellets have fully dissolved before adding more. It may take around 2 g(waay more) of NaOH pellets before the pH is at 8.0.
once close to 8 make a solution using pellets of naoh and slowly add the solution dropwise.
 
To make the TE buffer
Measure out 1 mL 1M Tris-Cl (pH 8.0) and add to a 100 mL beaker
Measure out 0.02 mL 0.5M EDTA (pH 8.0) and add to the beaker.
Top up the solution to 100 mL by adding distilled water.
Place the lid on the bottle and invert a few times to mix.
To sterilise, autoclave the solution on a liquid cycle (20 min at 15 psi).
 
Autoclaved everything and kept at room temperature:
  • The prepared TE buffer had a pH of 8.7

Tuesday - 7/11/2023

  • Resuspension of oligos:
    • We resuspended FRET aptamer cortisol (108288884), cDNA cortisol 1 (108288888), and cDNA cortisol 2 (108288889)
    • Oligo resuspension protocol:
        • First, briefly centrifuge the tubes before opening.
        • Resuspend in the prepared 1X TE buffer(10mM Tris HCl & 0.1mM EDTA), The tris Hcl acts as a buffer for the medium maintaining constant pH. The EDTA prevents the nuclear digestion of DNA.
        • Prepare the recommended 100μM stock.
        • If the aggregates are found after the addition of TE buffer, heat the mixture to 55 degree celsius for 1 to 5 mins
        • Vortex thoroughly then briefly centrifuge
        • If there are residual remains that are still found then run the mixture through Sephadex G-50 column
    • Using the above protocol, we prepared the stock of 100μM for each of the above mentioned oligos.
    • To prepare 10μM working concentration we used dilution calculator:
      • Add 10μl of 100μM stock concentration in 90μl of TE buffer solution to get a final volume of 100μl of 10μM working concentration
    • To prepare 0.1μM and 0.5μM aliquot of FRET aptamer cortisol
      • Add 2μl of 10μM working solution in 198μl of milliQ to get 200μl of 0.1 μM of FRET aptamer cortisol.
      • Add 10μl of 10μM working solution in 190μl of milliQ to get 200μl of 0.5 μM of FRET aptamer cortisol.
  • Measuring the fluorescence intensity of the 0.1μM and 0.5μM aliquots using a spectrofluorometer ( Fluorolog QM)
    • The FRET aptamer cortisol didn't show any considerable fluorescence for 0.1μM concentration, but a fluorescence peak was seen at 0.5μM concentration.
  • Fluorolog Protocol:
    • We have a 200μl cuvette .
    • First, we need to wash the cuvette. Wash the cuvette thoroughly first with milliQ, then with acetone and finally with ethanol.
    • Now, wash the cuvette with autoclaved Milli-Q once before taking 200μl MilliQ in the cuvette to take the base reading.
    • Wipe the cuvette with tissue and place it in the spectrofluorometer.
    • Set the protocol on the computer. We took a range scan to get emission readings between 495nm -700nm when the excitation peak was set at 495nm.
    • Run the protocol.
    • Empty the cuvette and wash thoroughly with MilliQ.
    • Add 200μl of sample in the cuvette. Wipe with a tissue and run the same protocol on the computer.
    • After taking all readings, wash the cuvette with acetone, ethanol and MilliQ thoroughly.

Wednesday - 7/12/2023

  • Resuspension of oligos:
    • We resuspended FRET aptamer cortisol (108288884), cDNA cortisol 1 (108288888), and cDNA cortisol 2 (108288889)
    • Oligo resuspension protocol:
        • First, briefly centrifuge the tubes before opening.
        • Resuspend in the prepared 1X TE buffer(10mM Tris HCl & 0.1mM EDTA), The tris HCl acts as a buffer for the medium maintaining constant pH. The EDTA prevents the nuclear digestion of DNA.
        • Prepare the recommended 100μM stock.
        • If the aggregates are found after the addition of TE buffer, heat the mixture to 55 degree celsius for 1 to 5 mins
        • Vortex thoroughly then briefly centrifuge
        • If there are residual remains that are still found then run the mixture through Sephadex G-50 column
    • Using the above protocol, we prepared the stock of 100μM for each of the above mentioned oligos.
    • To prepare 10μM working concentration we used dilution calculator:
      • Add 10μl of 100μM stock concentration in 90μl of TE buffer solution to get a final volume of 100μl of 10μM working concentration
    • To prepare 0.1μM and 0.5μM aliquot of FRET aptamer cortisol
      • Add 2μl of 10μM working solution in 198μl of milliQ to get 200μl of 0.1 μM of FRET aptamer cortisol.
      • Add 10μl of 10μM working solution in 190μl of milliQ to get 200μl of 0.5 μM of FRET aptamer cortisol.
  • Measuring the fluorescence intensity of the 0.1μM and 0.5μM aliquots using a spectrofluorometer ( Fluorolog QM)
    • The FRET aptamer cortisol didn't show any considerable fluorescence for 0.1μM concentration, but a fluorescence peak was seen at 0.5μM concentration.
  • Fluorolog Protocol:
    • We have a 200μl cuvette .
    • First, we need to wash the cuvette. Wash the cuvette thoroughly first with milliQ, then with acetone and finally with ethanol.
    • Now, wash the cuvette with autoclaved Milli-Q once before taking 200μl milliQ in the cuvette to take the base reading.
    • Wipe the cuvette with tissue and place it in the spectrofluorometer.
    • Set the protocol on the computer. We took a range scan to get emission readings between 495nm -700nm when the excitation peak was set at 495nm.
    • Run the protocol.
    • Empty the cuvette and wash thoroughly with MilliQ.
    • Add 200μl of sample in the cuvette. Wipe with a tissue and run the same protocol on the computer.
    • After taking all readings, wash the cuvette with acetone, ethanol and MilliQ thoroughly.

Thrusday - 7/13/2023

  • While measuring fluorescence intensity, we got a peak for 0.5 μM FRET aptamer cortisol. So next, we wanted to see if 0.25μM conc will also give a peak.
  • Made a 400μl 0.25μM FRET aptamer cortisol aliquot using 10μM working solution.
    • Take 10μl of 10μM FRET aptamer cortisol and add 390μl of MilliQ.
  • Measured the fluorescence intensity of 0.25μM FRET aptamer cortisol using fluorolog and got fluorescence peak.
  • Fluorolog Protocol:
    • Turn on the FluoroLog fluorometer and allow it to warm up for at least 15 minutes.
    • Attach the cuvette adaptor to the fluorometer.
    • We have a 200μl cuvette .
    • We need to wash the cuvette. Wash the cuvette thoroughly first with milliQ, then with acetone and finally with ethanol.
    • Now, wash the cuvette with autoclaved Milli-Q once before taking 200μl milliQ in the cuvette to take the base reading.
    • Wipe the cuvette with kim-tech wipes and place it in the spectrofluorometer.
    • Set the protocol on the computer. We took a range scan to get emission readings between 495nm -700nm when the excitation peak was set at 490nm.
    • Run the protocol.
    • Empty the cuvette and wash thoroughly with MilliQ.
    • Add 200μl of sample in the cuvette. Wipe with a tissue and run the same protocol on the computer.
    • After taking all readings, thoroughly wash the cuvette with acetone, ethanol and MilliQ.
    • Tips:
      • Make sure that the cuvette is clean and dry before using it.
      • Use a solvent that is compatible with your sample.
      • Fill the cuvette to the top to avoid bubbles.
      • Make sure that the cuvette is properly seated in the cuvette adaptor and the fluorometer.
      • Select the appropriate excitation and emission wavelengths for your sample.
      • Set the scan speed and step size to match the requirements of your sample.
      • Save the data to a computer for further analysis.
 
We decided to use 0.25μM FRET aptamer cortisol concentration constant for our experiments.

Friday - 7/14/2023

  • Incubation Time experiment for FRET aptamer cortisol and cDNA2 [1:1 concentration]- to check the optimal time required for FRET aptamer cortisol and cDNA2 to bind at specific concentrations.
  • Experiment Setup-
1) Take readings with 200μl MilliQ
2) Take 100μl 0.25μM FRET aptamer cortisol and 100μl MilliQ. Pipette mix it and take reading.
3) Take 100μl 0.25μM FRET aptamer cortisol and 100μl 0.25μM cDNA2. Pipette mix it and measure change in fluorescencee every 10 mins for 90 min.
  • Preparation of sample-
To make 200μl 0.25μM FRET aptamer cortisol, take 5μl of 10μM working concentration and 195μl MilliQ
To make 200μl 0.25μM cDNA2, take 5μl of 10μM working concentration and 195μl of MilliQ
  • Fluorolog Protocol:
      • We have a 200μl cuvette .
      • First, we need to wash the cuvette. Wash the cuvette thoroughly first with milliQ, then with acetone and finally with ethanol.
      • Now, wash the cuvette with autoclaved Milli-Q once before taking 200μl milliQ in the cuvette to take the base reading.
      • Wipe the cuvette with tissue and place it in the spectrofluorometer.
      • Set the protocol on the computer. We took a range scan to get emission readings between 495nm -700nm when the excitation peak was set at 495nm.
      • Run the protocol.
      • Empty the cuvette and wash thoroughly with MilliQ.
      • Add 200μl of sample in the cuvette. Wipe with a tissue and run the same protocol on the computer.
      • After taking all readings, wash the cuvette with acetone, ethanol and MilliQ thoroughly.
.
Thursday - 13/7/2023 Resuspension in TE buffer and dilution in water(autoclaved)
#LAF light off whenever using samples with fluorophore or quencher
miRNA 124-
  • SEQ A - 11.9 nm - 119ul TE
  • Seq- B - 38.3 - 383
  • Seq C - 31.7 - 317
  • miDNA 124 - 23.1 - 231
miRNA 132-
  • SEQ A - 15.8 - 158
  • Seq- B - 9.2 - 92
  • miDNA 132 - 30.1 -301
Aliquot of 10 uM concentration and 200 ul volume made:-
  • Take 20 ul of stock (100uM) and add 180 ul of autoclaved water.
  • Cover the sequences having fluorophore and/or quencher with aluminium foil, and store in -20 degrees.
Building probe for 124
#using 10uM aliquot
  • seq a = 15ul
  • seq b= 15 ul
  • seq c= 60 ul
  • final conc = 1.667 uM
  • final volume = 90 ul
  • label = 124 probe, date- 13/7/2023
  • Incubated at 48 degrees for 2 min and then at room temperature for 45 min.
  • store in 4 degrees.
Make a 1 uM sample of the probe to load
  • To get a 40 µL 1 µM solution, mix 24 µL of 1.667 µM oligo stock with 16 µL of water or TE buffer.
Making 2 uM solution for loading
  • To make 20 µL 2 µM solution, mix 4 µL of 10 µM oligo stock with 16 µL of water or TE buffer. Made for 124 A, B And C.
Making Native PAGE for DNA gel
  • 12 percent acrylamide bisacrylamide.
  • To make TBE (100 ml)
  • 10.8 g tris base
  • 5.5 g boric acid
  • 90 ml double-distilled H2O
  • 4 ml 0.5 M EDTA solution (pH 8.0)
  • To make poly acrylamide(19:1)
  • 19 g acrylamide
  • 1 g bisacrylamide
  • 80 ml water
  • stored in 4 degrees.
Friday - 14/7/2023
Making Native PAGE for DNA gel
  • 12 percent acrylamide bisacrylamide.
  • To make TBE (100 ml)
  • 10.8 g tris base
  • 5.5 g boric acid
  • 90 ml double-distilled H2O
  • 4 ml 0.5 M EDTA solution (pH 8.0)
  • To make poly acrylamide(19:1)
  • 19 g acrylamide
  • 1 g bisacrylamide
  • 80 ml water
  • stored in 4 degrees.
Need 4 gel
Each
 
  • Take 8 ml of the mixture.
  • add 0.5 percentthe volume of APS = 0.5 percent of 8 = .04ml aps= 40 ul 10 percent APS
  • TEMED- Take 1/10 of the volume of APS - 4ul (added 7)
add to a 15ml falcon , mix and pour fast.
wait 2 hour
 
Made miDNA 132 aliquot - 10uM
To get a 200 µL 10 µM solution, mix 20 µL of 100 µM oligo stock with 180 µL of water
 
Made 124 probe + miDNA
add 10ul 1uM probe and 2ul 10uM miDNA.
 
Made 132 probe + miDNA
add 13ul 1uM probe and 2ul 10uM miDNA.
To get a 30 µL 1 µM solution, mix 18 µL of 1.667 µM oligo stock with 12 µL of water or TE buffer.
 
Make A+B for 124 and 132
 
Add 8ul of 2uM A and B and incubate for 1 hour
 
Loading order- Labber (50bp), A, B, C, AB, probe, probe+miDNA
 
132- near glass
Run sample for 75 minutes at 55 v
Immerse the gel in syber safe for 30 min
store the used syber safe- labelled - used syber safe - in 50 ml flcon covered in aluminium foil
 
image
 


 
## inputs- use a 100 bp ladder just to show size
## remove gel and take image
 
 


 
##Increase conc of probe- load 15 ul each

Saturday - 7/15/2023

Procured Low range DNA marker (50μg) from MR lab courtesy of our mentor Tejas.

Sunday - 7/16/2023

  • NATIVE PAGE:
  • To run this Native gel we need to make the required concentration. of our oligos ie: the FRET aptamer and cDNA.
    • We decided to check the binding of the cDNA and aptamer, using the molar ratio of 1:3(aptamer : cDNA)
    • Sample prep
      • cDNA 2 conc-- 9 microliters of 10 micromolar stock solution 21 micromolar (milliQ) for forming 30 microliter 3micromolar
      • cDNA 2 conc-- 18 microliters of 10 micromolar stock solution 12 micromolar (milliQ) for forming 30 microliter 6micromolar
      • FRET aptamer conc-- 12 microliters of 10 micromolar stock solution 48 micromolar (milliQ) for forming 60 microliter 2micromolar
      • Incubate 30microlitre 2 micromolar FRET aptamer and 30 microlitre 6 micromolar cDNA for 60 min at 39°C to form the quantifier.
      • Divided the quantifier volume in half and added cortisol (13:2) to the quantifier and incubate again for 1 hour at 39°C.
      • Cortisol -- Made 0.2 mg/ml in 500 ml, then performed serial dilution to get 200ng/ml
Steps for serial dilution-- Label the tubes 1-7.
Add 100 microliters of the cortisol stock solution to tube 1.
Add 900 microliters of sterile water to tube 1.
Mix the contents of tube 1 thoroughly.
Transfer 100 microliters of the diluted cortisol solution from tube 1 to tube 2.
Add 900 microliters of sterile water to tube 2.
Mix the contents of tube 2 thoroughly.
Repeat steps 5-7 for tubes 3-6.
The final concentration of cortisol in tube 7 will be 200ng/ml.
      • Add 10microlitre loading dye for 30microlitre sample.
 
 
Preparation of 12 percent acrylamide bisacrylamide.
 
To make TBE (100 ml)
    • 10.8 g tris base
    • 5.5 g boric acid
    • 90 ml double-distilled H2O
    • 4 ml 0.5 M EDTA solution (pH 8.0)
heated a bit
 
To make poly acrylamide(19:1)
    • 19 g acrylamide
    • 1 g bisacrylamide
    • 80 ml water
stored in 4 degree (B1 ABL)

Friday - 7/14/2023

Making APS ( 10 percent)
    • Weight 1 g of ammonium persulphate
    • add about 9.5 ml of sterile ddH2O
    • Vortex until fully dissolved
    • store at 4°C.
Making gel mixture
    • 30 ml polyacrylamide(19:1)
    • 10ml 10x TBE
    • 60 ml milliQ
Making the gel
    • Take 8 ml of the mixture.
    • add 0.5 percent the volume of APS = 0.5 percent of 8 = .04ml APS = 40 ul 10 percent APS
    • TEMED- Take 1/10 of the volume of APS - 4ul (added 7ul)
    • Wipe apparatus with ethanol(glass plate)
    • setup the apparatus(SDS page apparatus-biorad miniprotein tetra)
    • check if theres leakage using water
    • if no, remove water and add mixture
    • wait 2 hour
    • shift the gel to the machine.
    • add 1x TBE load sample
sample prep -
    • 15ul sample + 3ul loading dye
    • vortex and spindown
 
Loading order-
Cortisol Quantifier+ Cortisol Quantifier FRET Aptamer Cortisol cDNA 2 cortisol
    • The gel was run at 55V for 1 hour( check after 1 hr).


    • We did not get results- our cDNA ran out, either we need to run it at a higher gel concentration or for a shorter duration of time.

Monday - 7/17/2023

  • Incubation Time experiment for FRET aptamer cortisol and cDNA2 [1:3 concentration]- to check the optimal time required for FRET aptamer cortisol and cDNA2 to bind at specific concentrations.
  • Experiment Setup-
1) Take readings with 200μl MilliQ
2) Take 100μl 0.25μM FRET aptamer cortisol and 100μl MilliQ. Pipette mix it and take reading.
3) Take 100μl 0.25μM FRET aptamer cortisol and 100μl 0.75μM cDNA2. Pipette mix it and measure change in fluorescencee every 10 mins for 90 min.
  • Preparation of sample-
To make 200μl 0.25μM FRET aptamer cortisol, take 100μl of 0.5μM working concentration and 100μl MilliQ
To make 200μl 0.75μM cDNA2, take 15μl of 10μM working concentration and 185μl of MilliQ
  • We received Serotonin, which is stored at 4°C.

Tuesday - 7/18/2023

  • Incubation Time experiment for FRET aptamer cortisol and cDNA2 [1:3 concentration] cont- to check the optimal time required for FRET aptamer cortisol and cDNA2 to bind at specific concentrations.
    • Experiment Setup-
1) Take readings with 200μl MilliQ
2) Take 100μl 0.25μM FRET aptamer cortisol and 100μl MilliQ. Pipette mix it and take reading.
3) Take 100μl 0.25μM FRET aptamer cortisol and 100μl 0.75μM cDNA2. Pipette mix it and measure the change in fluorescence every 10 mins for 90 min.
    • Preparation of sample- done on 7/17/2023
 
  • Incubation Time experiment for FRET aptamer cortisol and cDNA1 [1:3 concentration]- to check the optimal time required for FRET aptamer cortisol and cDNA1 to bind at specific concentrations.
      • Experiment Setup-
1) Take readings with 200μl MilliQ
2) Take 100μl 0.25μM FRET aptamer cortisol and 100μl MilliQ. Pipette mix it and take reading.
3) Take 100μl 0.25μM FRET aptamer cortisol and 100μl 0.75μM cDNA1. Pipette mix it and measure the change in fluorescence every 10 mins for 120 min.
      • Preparation of sample-
To make 200μl 0.25μM FRET aptamer cortisol, take 5μl of 10μM working concentration and 195μl MilliQ
To make 200μl 0.75μM cDNA1, take 15μl of 10μM working concentration and 185μl of MilliQ
  • Fluorolog Protocol:
          • We have a 200μl cuvette .
          • First, we need to wash the cuvette. Wash the cuvette thoroughly first with milliQ, then with acetone and finally with ethanol.
          • Now, wash the cuvette with autoclaved Milli-Q once before taking 200μl MilliQ in the cuvette to take the base reading.
          • Wipe the cuvette with tissue and place it in the spectrofluorometer.
          • Set the protocol on the computer. We took a range scan to get emission readings between 495nm -700nm when the absorbance peak was set at 495nm.
          • Run the protocol.
          • Empty the cuvette and wash thoroughly with MilliQ.
          • Add 200μl of sample in the cuvette. Wipe with a tissue and run the same protocol on the computer.
          • After taking all readings, wash the cuvette with acetone, ethanol and MilliQ thoroughly.
  • NATIVE PAGE [Repeat]
  • To run this Native gel we need to make the required concentration. of our oligos ie: the FRET aptamer and cDNA.
      • We decided to check the binding of the cDNA and aptamer, using the molar ratio of 1:3(aptamer : cDNA)
      • Sample prep
        • cDNA 2 conc-- 3 microliters of 10 micromolar stock solution 12 micromolar (milliQ) for forming 30 microliter 2micromolar
        • cDNA 2 conc-- 1 microliters of 100 micromolar stock solution 7 micromolar (milliQ) for forming 8 microliter 1micromolar
        • cDNA 1 conc-- 3 microliters of 10 micromolar stock solution 12 micromolar (milliQ) for forming 30 microliter 2micromolar
        • cDNA 1 conc-- 1 microliters of 100 micromolar stock solution 7 micromolar (milliQ) for forming 8 microliter 1micromolar
        • FRET aptamer conc-- 6 microliters of 10 micromolar stock solution 9 micromolar (milliQ) for forming 15 microliter 4micromolar
        • FRET aptamer conc-- 3 microliters of 10 micromolar stock solution 12 micromolar (milliQ) for forming 15 microliter 2micromolar
        • Incubate 30microlitre 2 micromolar FRET aptamer and 30 microlitre 6 micromolar cDNA for 60 min at 39°C to form the quantifier.
        • Divided the quantifier volume in half and added cortisol (13:2) to the quantifier and incubate again for 1 hour and 30 min at 39°C.
        • Cortisol -- Made 0.2 mg/ml in 500 ml, then performed serial dilution to get 200ng/ml
Steps for serial dilution-- Label the tubes 1-7.
Add 100 microliters of the cortisol stock solution to tube 1.
Add 900 microliters of sterile water to tube 1.
Mix the contents of tube 1 thoroughly.
Transfer 100 microliters of the diluted cortisol solution from tube 1 to tube 2.
Add 900 microliters of sterile water to tube 2.
Mix the contents of tube 2 thoroughly.
Repeat steps 5-7 for tubes 3-6.
The final concentration of cortisol in tube 7 will be 200ng/ml.
        • Add 10microlitre loading dye for 30microlitre sample.
 
 
Preparation of 12 percent acrylamide bisacrylamide.
 
To make TBE (100 ml)
      • 10.8 g tris base
      • 5.5 g boric acid
      • 90 ml double-distilled H2O
      • 4 ml 0.5 M EDTA solution (pH 8.0)
heated a bit
 
To make poly acrylamide(19:1)
      • 19 g acrylamide
      • 1 g bisacrylamide
      • 80 ml water
stored in 4 degree (B1 ABL)
 
Making APS ( 10 percent)
      • Weight 1 g of ammonium persulphate
      • add about 9.5 ml of sterile ddH2O
      • Vortex until fully dissolved
      • store at 4°C.
Making gel mixture
      • 30 ml polyacrylamide(19:1)
      • 10ml 10x TBE
      • 60 ml milliQ
Making the gel
      • Take 8 ml of the mixture.
      • add 0.5 percent the volume of APS = 0.5 percent of 8 = .04ml APS = 40 ul 10 percent APS
      • TEMED- Take 1/10 of the volume of APS - 4ul (added 7ul)
      • Wipe apparatus with ethanol(glass plate)
      • setup the apparatus(SDS page apparatus-biorad miniprotein tetra)
      • check if theres leakage using water
      • if no, remove water and add mixture
      • wait 2 hour
      • shift the gel to the machine.
      • add 1x TBE load sample
sample prep -
      • 15ul sample + 3ul loading dye
      • vortex and spindown
 
Loading order-
cDNA cortisol1 cDNA cortisol2 FRET aptamer cortisol FRET Quantifier 1 FRET Quantifier 2 Quantifier 1 with cortisol Quantifier 2 with cortisol
      • The gel was run at 55V for 1 hour ( check after 1 hr).
      • We did not get results again.

Friday - 7/21/2023

  • Incubation Time experiment for FRET aptamer cortisol and cDNA1 [1:2 concentration]- to check the optimal time required for FRET aptamer cortisol and cDNA1 to bind at specific concentrations.
  • Experiment Setup-
1) Take readings with 200μl MilliQ
2) Take 100μl 0.25μM FRET aptamer cortisol and 100μl MilliQ. Pipette mix it and take reading.
3) Take 100μl 0.25μM FRET aptamer cortisol and 100μl 0.5μM cDNA1. Pipette mix it and measure the change in fluorescence every 10 mins for 90 min.
  • Preparation of sample-
To make 200μl 0.25μM FRET aptamer cortisol, take 5μl of 10μM working concentration and 195μl MilliQ.
To make 200μl 0.5μM cDNA1, take 10μl of 10μM working concentration and 190μl of MilliQ
  • Fluorolog Protocol:
      • We have a 200μl cuvette .
      • First, we need to wash the cuvette. Wash the cuvette thoroughly first with milliQ, then with acetone and finally with ethanol.
      • Now, wash the cuvette with autoclaved Milli-Q once before taking 200μl milliQ in the cuvette to take the base reading.
      • Wipe the cuvette with tissue and place it in the spectrofluorometer.
      • Set the protocol on the computer. We took a range scan to get emission readings between 495nm -700nm when the excitation peak was set at 495nm.
      • Run the protocol.
      • Empty the cuvette and wash thoroughly with MilliQ.
      • Add 200μl of sample in the cuvette. Wipe with a tissue and run the same protocol on the computer.
      • After taking all readings, wash the cuvette with acetone, ethanol and MilliQ thoroughly.
.
Monday 7/17/2023
Make 124 probe
##using 10uM aliquot
  • seq a = 15ul
  • seq b= 15 ul
  • seq c= 60 ul
final conc = 1.667 uM
final volume = 90 ul
label = 132 probe 1.66uM, date
kept at 45 degree for 2 min and then at room temperature for 1 hour.
store in -4 degree.
 
Make A+B
##using 2uM aliquot
 
  • seq a = 10 ul
  • seq b= 10 ul
final conc = 1 uM
final volume = 20 ul
label = 124 AB, date
kept at 45 degree for 2 min and then at room temperature for 1 hour.
store in -4 degree.
 
Make 20ul of 2uM A and B
To get a 20 µL 2 µM solution, mix 4 µL of 10 µM oligo stock with 16 µL of water
Tuesday 7/18/2023
To make 1uM 124 probe
To get a 30 µL 1 µM solution, mix 18 µL of 1.667 µM oligo stock with 12 µL of water
 
Make 124 probe + miDNA
add 13ul 1uM probe and 2ul 10uM miDNA.
 
Making APS (10 percent)
  • Weight 0.1 g of ammonium persulphate
  • add about 0.95 ml of sterile ddH2O
  • Vortex until fully dissolved
  • store at 4°C.
 
Made gel
12 percent polyacrilamide solution- 8 ml
APS 10 percent- 40ul
TEMED - 7`ul
 
set for 2 hr
 
15ul sample- 3ul loading dye
run for 1 hour 55v
 
ladder used 100 BP
 
 


 
 
 
 
loading order- Ld, A,B,C,A+B, P,P+miDNA124.
Did not work
same result as the previous one.
Wednesday 7/19/2023
Page for 124 - trial 4 (increase concentration of probe)
Make probe + miDNA 124
15ul 1.66uM probe + 3ul 10uM miDNA
 
Make 2uM A and B seq
To get a 15 µL 2 µM solution, mix 3 µL of 10 µM oligo stock with 12 µL of water
 
Make A+B
##using 2uM aliquot
 
  • seq a = 10 ul
  • seq b= 10 ul
incubate for 2 min in 45 degree and then for 1 hour in room temperature.
 
Made gel
12 percent polyacrilamide solution- 8 ml
APS 10 percent- 40ul
TEMED - 7`ul
 
set for 2 hr
 
15ul sample- 3ul loading dye
Run for 1 hour 55v
 
ladder used 100 BP
loading order- Ld, A,B,C,A+B, P,P+miDNA124.
run for 1 hour 55v

Saturday - 7/22/2023

  • Incubation Time experiment for FRET aptamer cortisol and cDNA2 [1:2 concentration]- to check the optimal time required for FRET aptamer cortisol and cDNA2 to bind at specific concentrations.
  • Experiment Setup-
1) Take readings with 200μl MilliQ
2) Take 100μl 0.25μM FRET aptamer cortisol and 100μl MilliQ. Pipette mix it and take reading.
3) Take 100μl 0.25μM FRET aptamer cortisol and 100μl 0.5μM cDNA2. Pipette mix it and measure the change in fluorescence every 10 mins for 90 min.
  • Preparation of sample-
To make 200μl 0.25μM FRET aptamer cortisol, take 5μl of 10μM working concentration and 195μl MilliQ.
To make 200μl 0.5μM cDNA2, take 10μl of 10μM working concentration and 190μl of MilliQ
  • Fluorolog Protocol:
      • We have a 200μl cuvette .
      • First, we need to wash the cuvette. Wash the cuvette thoroughly first with milliQ, then with acetone and finally with ethanol.
      • Now, wash the cuvette with autoclaved Milli-Q once before taking 200μl milliQ in the cuvette to take the base reading.
      • Wipe the cuvette with tissue and place it in the spectrofluorometer.
      • Set the protocol on the computer. We took a range scan to get emission readings between 495nm -700nm when the excitation peak was set at 495nm.
      • Run the protocol.
      • Empty the cuvette and wash thoroughly with MilliQ.
      • Add 200μl of sample in the cuvette. Wipe with a tissue and run the same protocol on the computer.
      • After taking all readings, wash the cuvette with acetone, ethanol and MilliQ thoroughly.

Monday - 7/24/2023

  • Incubation Time experiment for FRET aptamer cortisol and cDNA2 and cDNA1 (1:4 concentration) - to check the optimal time required for FRET aptamer cortisol with cDNA2 and cDNA1 to bind at specific concentrations.
    • Experiment Setup-
1) Take readings with 200μl MilliQ
2) Take 100μl 0.25μM FRET aptamer cortisol and 100μl MilliQ. Pipette mix it and take reading.
3) Take 100μl 0.25μM FRET aptamer cortisol and 100μl 1μM cDNA2. Pipette mix it and measure the change in fluorescence every 10 mins for 90 min.
4)Take readings with 200μl MilliQ
5)Take 100μl 0.25μM FRET aptamer cortisol and 100μl MilliQ. Pipette mix it and take reading.
6)Take 100μl 0.25μM FRET aptamer cortisol and 100μl 1μM cDNA1. Pipette mix it and measure the change in fluorescence every 10 mins for 90 min.
  • Preparation of sample- [ for Incubation Time experiment for FRET aptamer cortisol and cDNA2 and cDNA1 (1:4 concentration) ]
To make 400μl 0.25μM FRET aptamer cortisol, take 10μl of 10μM working concentration and 390μl MilliQ.
To make 100μl 1μM cDNA1, take 10μl of 10μM working concentration and 90μl of MilliQ
To make 100μl 1μM cDNA2, take 10μl of 10μM working concentration and 90μl of MilliQ
  • Fluorolog Protocol:
        • We have a 200μl cuvette .
        • First, we need to wash the cuvette. Wash the cuvette thoroughly first with milliQ, then with acetone and finally with ethanol.
        • Now, wash the cuvette with autoclaved Milli-Q once before taking 200μl milliQ in the cuvette to take the base reading.
        • Wipe the cuvette with tissue and place it in the spectrofluorometer.
        • Set the protocol on the computer. We took a range scan to get emission readings between 495nm -700nm when the excitation peak was set at 495nm.
        • Run the protocol.
        • Empty the cuvette and wash thoroughly with MilliQ.
        • Add 200μl of sample in the cuvette. Wipe with a tissue and run the same protocol on the computer.
        • After taking all readings, wash the cuvette with acetone, ethanol and MilliQ thoroughly.
.

Sunday - 7/23/2023

P+miDNA
  • 20 ul 1.66 uM probe + 5ul miDNA
 
20ul 1.66 uM probe
 
30ul 2uM A and B
  • 30 µL 2 µM solution, mix 6 µL of 10 µM oligo stock with 24 µL
 
10ul C 10uM
 
A+B
  • 10ul 2uM A and B
  • 45 degree for 2 min and 1 hour in room temperature
 
Making gel
  • 30 ml polyacrylamide(19:1)
  • 10 ml 10x TBE
  • 60 ml milliQ
 
Making APS (10 percent)
  • Weight 0.1 g of ammonium persulphate
  • add about 0.95 ml of sterile ddH2O
  • Vortex until fully dissolved
  • store at 4°C.
 
make gel - 40ul aps, 7ul temed
 
Add 3ul loading dye to all ,and load
55v 1 hour
ladder10, a, b, c, ab, p ,p+mi
 


Monday - 7/24/2023


 
0.1 g- 1ml- very clay like,
so 0.1 g in 10 ml(make upto 10ml)
0.01g/ml
 
Take 100ul and add 900ul water
0.001= 1 milligram/ml
 
Take 500ul add 500ul water
0.5mg/ml

Monday - 8/4/2023

  • Autoclaved MilliQ
  • We did resuspension of the following primers using TE buffer-
1) forward primer (108288891) - add 235μl of TE buffer
2) reverse primer (108288892) - add 296μl of TE buffer
3) gsα - we have 1000ng/μl, to make 10ng/μl, we add 100μl of TE buffer.
  • Same Resuspension protocol was used.
    • Oligo resuspension protocol:
          • First, briefly centrifuge the tubes before opening.
          • Resuspend in the prepared 1X TE buffer(10mM Tris HCl & 0.1mM EDTA), The tris HCl acts as a buffer for the medium maintaining constant pH. The EDTA prevents the nuclear digestion of DNA.
          • Prepare the recommended 100μM stock.
          • If the aggregates are found after the addition of TE buffer, heat the mixture to 55 degree celsius for 1 to 5 mins
          • Vortex thoroughly then briefly centrifuge
          • If there are residual remains that are still found then run the mixture through Sephadex G-50 column
.

Friday - 8/11/2023

PLATE READER STUFF

 
use water- 107 ul
100ul 0.5um probe +7um water
100ul 0.5um probe +7um midna
 
200 µL 0.25 µM solution, mix 30.1 µL of 1.66 µM oligo stock with 169.9 µL
 
12 water
1a - water
1b - pbr
1c prb+dna
 
 
Make 124 probe
##using 10uM aliquot
 
  • seq a = 15ul
  • seq b= 15 ul
  • seq c= 60 ul
 
final conc = 1.667 uM
final volume = 90 ul
label = 124 probe 1.66uM, date
kept at 45 degree for 2 min and then at room temperature for 1 hour.
store in -4 degree.
 
Make 132 probe
##using 10uM aliquot
 
  • seq a = 15ul
  • seq b= 15 ul
  • seq c= 60 ul`
 
final conc = 1.667 uM
final volume = 90 ul
label = 132 probe 1.66uM, date
kept at 45 degree for 2 min and then at room temperature for 1 hour.
store in -4 degree.
To get a 200 µL 0.25 µM solution, mix 30.1 µL of 1.66 µM oligo stock with 169.9 µL of water.

Monday - 8/7/2023

9:30 am - Autoclaved Nuclease Free water

Wednesday - 8/9/2023

RESUSPENSION OF DNA AND PRIMERS
  • To make 10ng/ul stock solution of gsα gene fragment, added 100 ul nuclease free water to TWIST tube containing 1000ng dried down DNA. Spin down, vortexed and stored in -20degree iGEM stock box
  • To resuspend forward primers (23.5 nmol) - added 235 ul TE buffer. Spin down, vortexed and stored in -20 degree stock box. Final concentration - 100 uM
  • To resuspend backward primers (29.6 nmol) - added 296 ul TE buffer. Spin down, vortexed and stored in -20 degree stock box. Final concentration - 100 uM

Friday - 8/11/2023

PREPARATION OF WORKING STOCK
  • To make working stock of 100 ul of 0.2uM, added 0.2 uL of Forward primer stock to 99.8 uL milliQ
  • To make working stock of 100 ul of 0.2uM, added 0.2 uL of Reverse primer stock to 99.8 uL milliQ
  • To make working stock of 50 uL of 200 uM, added 0.25 ul of 10mM dNTPs to 49.75 ul milliQ

Monday - 8/5/2023

  • We did Resuspension of the following oligos to prepare 100μM stock concentration.
1) FRET aptamer cortisol truncated- add 161μl of TE buffer.
2)cDNA truncated cortisol- add 383μl of TE buffer
  • Same Resuspension protocol was used.
Oligo resuspension protocol:
        • First, briefly centrifuge the tubes before opening.
        • Resuspend in the prepared 1X TE buffer(10mM Tris HCl & 0.1mM EDTA), The tris HCl acts as a buffer for the medium maintaining constant pH. The EDTA prevents the nuclear digestion of DNA.
        • Prepare the recommended 100μM stock.
        • If the aggregates are found after the addition of TE buffer, heat the mixture to 55 degree celsius for 1 to 5 mins
        • Vortex thoroughly then briefly centrifuge
        • If there are residual remains that are still found, then run the mixture through Sephadex G-50 column
  • We then prepared sample by performing dilutions using MilliQ.
1) To make 200μl 10μM FRET aptamer cortisol truncated, add 20μl of 100μM oligo and 180μl of MilliQ.
2) To make 200μl 0.25μM FRET aptamer cortisol truncated, add 5μl of 10μM oligo and 195μl of MilliQ.
3)To make 200μl 10μM cDNA truncated cortisol, add 20μl of 100μM oligo and 180μl of MilliQ.
4)To make 200μl 0.5μM cDNA cortisol truncated, add 10μl of 10μM oligo and 190μl of MilliQ.
  • Incubation Time experiment for FRET aptamer cortisol truncated and cDNA truncated (1:2concentration) - to check the optimal time required for FRET aptamer cortisol truncated with cDNA trn to bind at specific concentrations.
  • Experimental setup-
1) Take readings with 200μl MilliQ
2) Take 100μl 0.25μM FRET aptamer cortisol truncated and 100μl MilliQ. Pipette mix it and take reading.
3) Take 100μl 0.25μM FRET aptamer truncated cortisol and 100μl 0.5μM cDNA truncated cortisol. Pipette mix it and measure the change in fluorescence every 10 mins for 90 min.
  • Fluorolog Protocol:
        • We have a 200μl cuvette .
        • First, we need to wash the cuvette. Wash the cuvette thoroughly first with milliQ, then with acetone and finally with ethanol.
        • Now, wash the cuvette with autoclaved Milli-Q once before taking 200μl milliQ in the cuvette to take the base reading.
        • Wipe the cuvette with tissue and place it in the spectrofluorometer.
        • Set the protocol on the computer. We took a range scan to get emission readings between 495nm -700nm when the excitation peak was set at 495nm.
        • Run the protocol.
        • Empty the cuvette and wash thoroughly with MilliQ.
        • Add 200μl of sample in the cuvette. Wipe with a tissue and run the same protocol on the computer.
        • After taking all readings, wash the cuvette with acetone, ethanol and MilliQ thoroughly.

Thursday - 8/10/2023

  • Incubation Time experiment for FRET aptamer cortisol truncated and cDNA truncated (1:5concentration) - to check the optimal time required for FRET aptamer cortisol truncated with cDNA trn to bind at specific concentrations.
  • Preparation of sample-
    • For FRET aptamertruncated 0.5 micro molar , add 20 microlitre of 10 micro molar stock in 190 microlitre milliQ to get 200microlitre sample.
    • For cdna truncated 2.5 micro molar , add 50 microlitre of 10 micro molar stock in 150 microlitre milliQ to get 200microlitre sample.
  • Experimental setup-
1) Take readings with 200μl MilliQ
2) Take 100μl 0.5μM FRET aptamer cortisol truncated and 100μl MilliQ. Pipette mix it and take reading.
3) Take 100μl 0.5μM FRET aptamer truncated cortisol and 100μl 2.5μM cDNA truncated cortisol. Pipette mix it and measure the change in fluorescence every 10 mins for 90 min.
  • Fluorolog Protocol:
          • We have a 200μl cuvette .
          • First, we need to wash the cuvette. Wash the cuvette thoroughly first with milliQ, then with acetone and finally with ethanol.
          • Now, wash the cuvette with autoclaved Milli-Q once before taking 200μl MilliQ in the cuvette to take the base reading.
          • Wipe the cuvette with tissue and place it in the spectrofluorometer.
          • Set the protocol on the computer. We took a range scan to get emission readings between 495nm -700nm when the excitation peak was set at 495nm.
          • Run the protocol.
          • Empty the cuvette and wash thoroughly with MilliQ.
          • Add 200μl of sample in the cuvette. Wipe with a tissue and run the same protocol on the computer.
          • After taking all readings, wash the cuvette with acetone, ethanol and MilliQ thoroughly.

Friday - 8/11/2023

  • Incubation Time experiment for FRET aptamer cortisol and cDNA 1 (1:2 concentration) - to check the optimal time required for FRET aptamer cortisol with cDNA 1 to bind at specific concentrations.
  • Preparation of sample-
      • For FRET aptamer cortisol 0.25 micro molar , add 5 microlitre of 10 micro molar stock in 195 microlitre milliQ to get 200microlitre sample.
      • For cdna 1 cortisol 0.5 micro molar , add 10 microlitre of 10 micro molar stock in 190 microlitre milliQ to get 200microlitre sample.
  • Experimental setup-
1) Take readings with 200μl MilliQ
2) Take 100μl 0.25μM FRET aptamer cortisol and 100μl MilliQ. Pipette mix it and take reading.
3) Take 100μl 0.25μM FRET aptamer cortisol and 100μl 0.5μM cDNA1 cortisol. Pipette mix it and measure the change in fluorescence every 10 mins for 90 min.
  • Fluorolog Protocol:
          • We have a 200μl cuvette .
          • First, we need to wash the cuvette. Wash the cuvette thoroughly first with milliQ, then with acetone and finally with ethanol.
          • Now, wash the cuvette with autoclaved Milli-Q once before taking 200μl MilliQ in the cuvette to take the base reading.
          • Wipe the cuvette with tissue and place it in the spectrofluorometer.
          • Set the protocol on the computer. We took a range scan to get emission readings between 495nm -700nm when the excitation peak was set at 495nm.
          • Run the protocol.
          • Empty the cuvette and wash thoroughly with MilliQ.
          • Add 200μl of sample in the cuvette. Wipe with a tissue and run the same protocol on the computer.
          • After taking all readings, wash the cuvette with acetone, ethanol and MilliQ thoroughly.

Saturday - 8/12/2023

PCR AMPLIFICATION OF gsα GBLOCK
  Fnal concentration Volume added
NEB Buffer   2.5 uL
dNTPSs 200uM 0.5 uL
Forward primer 0.2uM 0.5 uL
Reverse primer 0.2uM 0.5 uL
Template DNA 10ng/uL 0.2 uL
NEB Taq polymerase   0.125 uL
Nuclease free water   20.675 uL
Total volume   25 uL
PCR protocol -saved under IGEMGSAP - ABL Thermocycler
 


Annealing temp - 57 degrees

Sunday - 8/13/2023

AGAROSE GEL ELECTROPHORESIS
To make a 2 percent gel (70 mL);
  • 7 uL 10 X TBE in 63 uL milliQ for 60 mL 1X TBE
  • Added 1.4 g low EEO agarose
  • Melt for 1 min 30 secs, streaked with 2 uL EtBr, left to solidify for 45 mins
  • Loaded 25uL sample + 5 uL loading dye
  • Used two 1 kb ladder -
2.5 uL NEB ladder + 2 uL purple loading dye
5 ul Origin ladder (purple colour)
  • Ran gel at 375 mA and 80 volts for 1 hour 30 mins
Gel imaged in the Gel Doc system - Filter 1
Saved under volume D > iGEM > iGEM 23 >GSAP pcr amp 1
 
gsap pcr amp 1.scn
No bands were observed.
 
PCR AMPLIFICATION 2.0
 
Used lab grade Taq polymerase and buffer from Satish lab
Buffer   2.5 uL
dNTPSs 200uM 0.5 uL
Forward primer 0.2uM 0.5 uL
Reverse primer 0.2uM 0.5 uL
Template DNA 10ng/uL 0.2 uL
Taq polymerase   0.5 uL
Nuclease free water   22.8 uL
Total volume   25 uL
6:30 pm - Same PCR protocols in ABL Thermocycler
Ran for 2 h 18 mins
Stored in 4 degree

Monday - 8/14/2023

AGAR GEL 2.0
  • Made 60 mL1x TBE buffer - 6mL TBE + 54 mL milliQ
  • 0.6g low EEO agarose
  • Melt for 1 min 30 secs, streaked with 2 uL EtBr, left to solidify for 45 mins
  • Loaded 25uL sample + 5 uL loading dye
  • 2.5 uL NEB ladder + 2 uL purple loading dye
  • Ran gel at 375 mA and 80 volts for 1 hour 30 mins
NO BANDS OBSERVED.

Wednesday - 8/16/2023

Positive control (1.5kB) and primers procured from MR lab.
Annealing temp- 57 degrees
 
GRADIENT PCR AMP 1.0
Since discrepencies in calculated and given melting temperature
Made 5 tubes acc to PCR mixture 2.0 using lab grade Taq polymerase and buffer from MR lab labelled C,D,E,F,G
Made 2 tubes for positive control
  • checked con
parts Final con Positive control A Positive control B C D E F G
Buffer   1.25 uL 2.5uL 2.5 uL 2.5 uL 2.5 uL 2.5 uL 2.5 uL
dNTPSs 200uM 0.125 uL 0.5uL 0.5 uL 0.5 uL 0.5 uL 0.5 uL 0.5 uL
Forward primer 0.2uM 0.125 uL 0.5uL 0.5 uL 0.5 uL 0.5 uL 0.5 uL 0.5 uL
Reverse primer 0.2uM 0.125 uL 0.5uL 0.5 uL 0.5 uL 0.5 uL 0.5 uL 0.5 uL
Template DNA 10ng/uL 0.2 uL 0.2 0.2 uL 0.2 uL 0.2 uL 0.2 uL 0.2 uL
Taq polymerase   0.125 uL 0.5uL 0.5 uL 0.5 uL 0.5 uL 0.5 uL 0.5 uL
Nuclease free water   13.05 uL 22.8 uL 22.8 uL 22.8 uL 22.8 uL 22.8 uL 22.8 uL
Total volume   15 uL 25 uL 25 uL 25 uL 25 uL 25 uL 25 uL
Annealing temp   57.1 57.1 57.1 58 61.2 62.5 63.1
 
GRADIENT PCR 1.1



Thursday - 8/17/2023

GRADIENT PCR 1.1
Added polymerase didn't have Mgcl2 - faulty polymerase.
After completing gradient pcr tried adding Mgcl2 to each tubes
Conc of Mgcl2 we had=25mM
Required conc of Mgcl2= 1.5mM
Quantity of each sample=25uL
To each tube( having 25uL sample) add:
  • 1.8uL of mgcl2
  • 3.2uL of nuclease free water.
Hence took 9uL Mgcl2(5 * 1.8) and 16uL nuclease free water(5 * 3.2), mixed them and added 5uL each in tubes B,C,D,E,F,G
To Sample A( Quantity= 15uL), add:
  • .3uL Mgcl2
  • 4.7uL nuclease free water
Ran gradient PCR again ( Temparature range-Gradient PCR 1.0)
Ran gel again
NO BANDS.( Adding Mgcl2 after PCR didn't work)

Friday - 8/18/2023

GRADIENT PCR 2.0
Taq polymerase with MgCl2 pre- added
Parts con Positive control H I J K L
Buffer   2.5 uL 2.5 uL 2.5 uL 2.5 uL 2.5 uL 2.5 uL
dNTPSs 200uM 0.5 uL 0.5 uL 0.5 uL 0.5 uL 0.5 uL 0.5 uL
Forward primer 0.2uM 0.5 uL 0.5 uL 0.5 uL 0.5 uL 0.5 uL 0.5 uL
Reverse primer 0.2uM 0.5 uL 0.5 uL 0.5 uL 0.5 uL 0.5 uL 0.5 uL
Template DNA 10ng/uL 0.5uL 0.5uL 0.5uL 0.5uL 0.5uL 0.5uL
Taq polymerase   0.5 uL 0.5 uL 0.5 uL 0.5 uL 0.5 uL 0.5 uL
Nuclease free water   22.8 uL 22.8 uL 22.8 uL 22.8 uL 22.8 uL 22.8 uL
Total volume   25 uL 25 uL 25 uL 25 uL 25 uL 25 uL
Annealing temp   57.1 57.1 58 61.2 62.5 63.1
Same Mastercycler protocols as Gradient PCR 1.0
In addition, ran an extra PCR with the positive in ABL Thermocycler
 
parts Con M
Buffer   2.5 uL
dNTPSs 200uM 1 uL
Forward primer 0.2uM 0.5 uL
Reverse primer 0.2uM 0.5 uL
Template DNA 10ng/uL 0.5uL
Taq polymerase   0.5 uL
Nuclease free water   19.5uL
Total volume   25 uL
Annealing temp   57


Ran AGAR GEL

Sunday - 8/13/2023

  • Incubation Time experiment for FRET aptamer cortisol and cDNA 1 (1:3 concentration) - to check the optimal time required for FRET aptamer cortisol with cDNA 1 to bind at specific concentrations.
  • Preparation of sample-
    • For FRET aptamer0.25 micro molar , add 5 microlitre of 10 micro molar stock in 195 microlitre milliQ to get 200microlitre sample.
    • For cdna 1 0.75 micro molar , add 15 microlitre of 10 micro molar stock in 185 microlitre milliQ to get 200 microlitre sample.
  • Experimental setup-
1) Take readings with 200μl MilliQ
2) Take 100μl 0.25μM FRET aptamer cortisol and 100μl MilliQ. Pipette mix it and take reading.
3) Take 100μl 0.25μM FRET aptamer cortisol and 100μl 0.75μM cDNA1 cortisol. Pipette mix it and measure the change in fluorescence every 10 mins for 90 min.
    • Fluorolog Protocol:
          • We have a 200μl cuvette .
          • First, we need to wash the cuvette. Wash the cuvette thoroughly first with milliQ, then with acetone and finally with ethanol.
          • Now, wash the cuvette with autoclaved Milli-Q once before taking 200μl milliQ in the cuvette to take the base reading.
          • Wipe the cuvette with tissue and place it in the spectrofluorometer.
          • Set the protocol on the computer. We took a range scan to get emission readings between 495nm -700nm when the excitation peak was set at 495nm.
          • Run the protocol.
          • Empty the cuvette and wash thoroughly with MilliQ.
          • Add 200μl of sample in the cuvette. Wipe with a tissue and run the same protocol on the computer.
          • After taking all readings, wash the cuvette with acetone, ethanol and MilliQ thoroughly.

Saturday - 8/19/2023

Ran AGAR GEL
  • Made 60 mL1x TBE buffer - 6mL TBE + 54 mL milliQ
  • 0.6g low EEO agarose
  • Melt for 1 min 30 secs, streaked with 2 uL EtBr, left to solidify for 45 mins
  • Loaded 25uL sample + 5 uL loading dye
  • 2.5 uL NEB ladder + 2 uL purple loading dye
  • Ran gel at 375 mA and 80 volts for 1 hour 30 mins
WE GOT THE BANDS
 
 
 



 
 
 
ELUTION
  • Preheat heat block in 50 degrees
  • Cut the gel and weigh it . For every 100mg take 200uL NT-1 ( Binding buffer )
Weight of gel= 600mg
NT-1 Buffer taken= 1200uL , in 1.5ml tube.
  • Melted the gel (approx 15 min). Vortex it in every 5 min
  • Took 2ml collecting tube and coloumn.Added 700uL of melted gel through sides of tube.
  • Centrifuge it , 1000 rpm for 3 min
  • After 3 min poured the solution in collecting tube back to coloumn
Repeat this thrice.
  • After repeating thrice , stored the buffer in another tube in 4 degrees.
  • Took 600uL wash buffer( NT-3) and added it into coloumn. Kept it undisturbed for 3 min
  • After 3 min ,Centrifuge it in 2000rpm for 3 min
  • After centrifuging discarded the wash buffer and repeated the step once more
  • Then kept the tube in 13k rpm for 2 min - Dry spin
  • Preheat 20uL of elution buffer in 50 degree
  • Took 1.5ml autoclaved tube and added 10uL of elution buffer to coloumn
  • Centrifuge it in 13k rpm for 2 min
  • Added the remaining 10uL elution buffer to coloumn and centrifuge it in 13k rpm for 2 min
 
NANODROP
Got a conc of 6.1 ng/uL

Monday - 8/21/2023

PCR AMPLIFICATION 3.0
Did a final pcr
  Final concentration     Volume added
NEB Buffer   2.5 2.5 2.5
 dNTPs 200uM 1.5 1.5 1.5
Forward primer 0.2uM 0.5 0.5 0.5
Reverse primer 0.2uM 0.5 0.5 0.5
Template DNA 10ng/uL 0.5 0.5 0.5
NEB Taq polymerase   0.5 0.5 0.5
Nuclease free water        
Total volume   25 uL 25 uL 25 uL

Tuesday - 8/22/2023

Made 500mL TBE buffer stock from 50X TBE
 
Ran AGAR GEL
  • Made 60 mL1x TBE buffer - 6mL TBE + 54 mL milliQ
  • 0.6g low EEO agarose
  • Melt for 1 min 30 secs, streaked with 2 uL EtBr, left to solidify for 45 mins
  • Loaded 25uL sample + 5 uL loading dye
  • 2.5 uL NEB ladder + 2 uL purple loading dye
  • Ran gel at 375 mA and 80 volts for 1 hour 30 mins


 
 
ELUTION 1.0
  • Preheat heat block in 50 degrees
  • Cut the gel and weigh it . For every 100mg take 200uL NT-1 ( Binding buffer )
Weight of gel= 406mg
 
NT-1 Buffer taken= 800uL , in 1.5ml tube.
  • Melted the gel (approx 15 min). Vortex it in every 5 min
  • Took 2ml collecting tube and coloumn.Added 700uL of melted gel through sides of tube.
  • Centrifuge it , 1000 rpm for 3 min
  • After 3 min poured the solution in collecting tube back to coloumn
Repeat this thrice.
  • After repeating thrice , stored the buffer in another tube (Tube-1) in 4 degrees.
  • Repeated the same steps with remaining NT-1 buffer( of 800uL)
Stored buffer in Tube-1 in 4 degrees.
  • Took 600uL wash buffer( NT-3) and added it into coloumn. Kept it undisturbed for 3 min
  • After 3 min ,Centrifuge it in 2000rpm for 3 min
  • After centrifuging discarded the wash buffer and repeated the step once more
  • Then kept the tube in 13k rpm for 2 min - Dry spin
  • Preheat 20uL of elution buffer in 50 degree
  • Took 1.5ml autoclaved tube and added 10uL of elution buffer to coloumn
  • Centrifuge it in 13k rpm for 2 min
  • Added the remaining 10uL elution buffer to coloumn and centrifuge it in 13k rpm for 2 min

Wednesday - 8/23/2023

NANODROP
Got a conc of 8.1 ng/uL
RESTRICTION DIGESTION
Restriction mixture
R1 containing DNA insert - Amplified gsα of 8.1ng / uL con
2. R2 containing Vector - pET19b 128ng/uL
 
  R1 R2
To be cut 15 uL- 121ng  
Cutsmart buffer 2 uL 2 uL
BamH1-HF 0.2 uL 0.2 uL
Nde1 HF 0.3 uL 0.3 uL
Nuclease free water 2.5 uL 2.5 uL
Total volume 20 uL 20 uL
Restriction enzymes;
  • Keep on ice when not in the freezer - minimize exposure to temp above -20degree
  • Should be the last component added to reaction
  • Mix components by pipetting the reaction mixture up and down, or by "flicking" the reaction tube. Follow with a quick ("touch") spin-down in a microcentrifuge. Do not vortex the reaction
Incubated at 37 degree overnight
Stored in -20 degrees post incubation

Thursday- 31/8/2023

Making 0.5mg/ml solution of magnetic particle

 Take 100ul and add 900ul water

0.001= 1 milligram/ml

 Take 500ul add 500ul water

0.5mg/ml

  TEM sample preparation (new batch)

 Take 100 ul 0.5 mg/ml nanoparticle and add 400 ul 20 percent ethanol-water mixture.

take 6 u from this and drop it on a copper grid.

Let it dry in the open

Once dried, store in an Eppendorf tube.

Friday - 1/9/2023

  TEM ANALYSIS

  Particle size still low but chose to continue with this.

 

Sunday - 8/27/2023

  • Native page - repeat
  • To run this Native gel we need to make the required concentration. of our oligos ie: the FRET aptamer and cDNA.
      • We decided to check the binding of the cDNA and aptamer, using the molar ratio of 1:3(aptamer : cDNA)
      • Sample prep
        • cDNA 1 conc-- 9 microliters of 10 micromolar stock solution 21 micromolar (milliQ) for forming 30 microliter 3micromolar
        • cDNA 1 conc-- 18 microliters of 10 micromolar stock solution 12 micromolar (milliQ) for forming 30 microliter 6micromolar
        • FRET aptamer conc-- 12 microliters of 10 micromolar stock solution 48 micromolar (milliQ) for forming 60 microliter 2micromolar
        • FRET aptamer conc-- 24 microliters of 10 micromolar stock solution 36 micromolar (milliQ) for forming 60 microliter 4micromolar
        • Incubate 30microlitre 2 micromolar FRET aptamer and 30 microlitre 6 micromolar cDNA for 60 min at 39°C to form the quantifier.
        • Divided the quantifier volume in half and added cortisol (13:2) to the quantifier and incubate again for 1 hour at 39°C.
        • Cortisol -- Made 0.2 mg/ml in 500 ml, then performed serial dilution to get 200ng/ml
Steps for serial dilution-- Label the tubes 1-7.
Add 100 microliters of the cortisol stock solution to tube 1.
Add 900 microliters of sterile water to tube 1.
Mix the contents of tube 1 thoroughly.
Transfer 100 microliters of the diluted cortisol solution from tube 1 to tube 2.
Add 900 microliters of sterile water to tube 2.
Mix the contents of tube 2 thoroughly.
Repeat steps 5-7 for tubes 3-6.
The final concentration of cortisol in tube 7 will be 200ng/ml.
        • Add 10microlitre loading dye for 30microlitre sample.
 
 
Preparation of 12 percent acrylamide bisacrylamide.
 
To make TBE (100 ml)
      • 10.8 g tris base
      • 5.5 g boric acid
      • 90 ml double-distilled H2O
      • 4 ml 0.5 M EDTA solution (pH 8.0)
heated a bit
 
To make poly acrylamide(19:1)
      • 19 g acrylamide
      • 1 g bisacrylamide
      • 80 ml water
stored in 4 degree (B1 ABL)
 
Making APS ( 10 percent)
      • Weight 1 g of ammonium persulphate
      • add about 9.5 ml of sterile ddH2O
      • Vortex until fully dissolved
      • store at 4°C.
Making gel mixture
      • 30 ml polyacrylamide(19:1)
      • 10ml 10x TBE
      • 60 ml milliQ
Making the gel
      • Take 8 ml of the mixture.
      • add 0.5 percent the volume of APS = 0.5 percent of 8 = .04ml APS = 40 ul 10 percent APS
      • TEMED- Take 1/10 of the volume of APS - 4ul (added 7ul)
      • Wipe apparatus with ethanol(glass plate)
      • setup the apparatus(SDS page apparatus-biorad miniprotein tetra)
      • check if theres leakage using water
      • if no, remove water and add mixture
      • wait 2 hour
      • shift the gel to the machine.
      • add 1x TBE load sample
sample prep -
      • 15ul sample + 3ul loading dye
      • vortex and spindown
 
Loading order-
ladder FRET Aptamer cDNA 1 cortisol quantifier quantifier +cortisol
      • The gel was run at 55V for 1 hour( check after 1 hr).
      • we still did not get good results, cdna ran out again.

Tuesday - 9/5/2023

making MES buffer (15 mM)

 Measure 0.319g MES monohydrate

make upto 100 ml

 Adjust ph to 6 using 10 N NaOH (fast change in pH)(6.1)

 store in -20

10 N NaOH - Mix 4g pellet in 8 ml water.

Friday - 9/8/2023

Running DLS - new batch

To run DLS take 50 ul 0.5 mg nanoparticle solution and add 450 ul and vortex

Add the sample to the cuvette and run.

 

-21 mV - stable carboxyl groups proven

Monday - 9/4/2023

RESTRICTION DIGESTION
Restriction mixture
R1 containing DNA insert - Amplified gsα of 8.1ng / uL con
2. R2 containing Vector - pET19b 128ng/uL
 
  R1 R2
To be cut 15 uL- 121ng  
Cutsmart buffer 2 uL 2 uL
BamH1-HF 0.2 uL 0.2 uL
Nde1 HF 0.3 uL 0.3 uL
Nuclease free water 2.5 uL 2.5 uL
Total volume 20 uL 20 uL
Restriction enzymes;
  • Keep on ice when not in the freezer - minimize exposure to temp above -20degree
  • Should be the last component added to reaction
  • Mix components by pipetting the reaction mixture up and down, or by "flicking" the reaction tube. Follow with a quick ("touch") spin-down in a microcentrifuge. Do not vortex the reaction
 
Incubated at 37 degree overnight
Stored in -20 degrees post incubation

Tuesday - 9/5/2023

Ran AGAR gel
  • Made 60 mL1x TBE buffer - 6mL TBE + 54 mL milliQ
  • 0.6g low EEO agarose
  • Melt for 1 min 30 secs, streaked with 2 uL EtBr, left to solidify for 45 mins
  • Loaded 25uL sample + 5 uL loading dye
  • 2.5 uL NEB ladder + 2 uL purple loading dye
  • Ran gel at 375 mA and 80 volts for 1 hour 30 mins 
 
Band for plasmid was visible
Band for insert wasn't visible- INSERT CON INSUFFICIENT.
 
ELUTION (Plasmid)
Elution 1.0
Weight of gel= 260mg
Amount of NT 1 added=500uL
 
NANODROP(Plasmid)
Got a conc of 8ng/uL
 
PCR
To amplify insert
Optimizing for high yield - increasing primer concentration, inreasing number of cycles
 
Buffer   2.5 uL 2.5 uL 2.5 uL
dNTPSs 200uM 1 uL 1 uL 1 uL
Forward primer 0.2uM 1 uL 1 uL 1 uL
Reverse primer 0.2uM 1 uL 1 uL 1 uL
Template DNA 10ng/uL 0.5 uL 0.5 uL 0.5 uL
Taq polymerase   0.5 uL 0.5 uL 0.5 uL
Nuclease free water   18.5 uL 18.5 uL 18.5 uL
Total volume   25 uL 25 uL 25 uL
BAND NOT VISIBLE

Wednesday - 9/6/2023

AMPLIFICATION PCR
To amplify insert
Buffer   2.5uL    
dNTPSs 200uM 1.5 uL    
Forward primer 0.2uM 0.5    
Reverse primer 0.2uM 0.5    
Template DNA 10ng/uL 1uL    
Taq polymerase   0.5 uL    
Nuclease free water   18.5uL    
Total volume   25 uL    
Ran AGAR gel
  • Made 50 mL 1x TBE buffer - 5mL TBE + 45 mL milliQ
  • 0.5g low EEO agarose
  • Melt for 1 min 30 secs, streaked with 2 uL EtBr, left to solidify for 45 mins
  • Loaded 25uL sample + 5 uL loading dye
  • 2.5 uL NEB ladder + 2 uL purple loading dye
  • Ran gel at 375 mA and 80 volts for 1 hour 30 mins
 

Thursday - 9/7/2023

ELUTION (insert)
    • Elution 1.0 done
    • Weight of gel - 253mg
    • Amount of NT1 added 600uL
NANODROP
con -4.2 ng/uL
 
LIGATION
 
  Conc L1
Inser 4.2 ng/uL 4.5uL
Vector 8ng/uL 8uL
T4 ligase   1uL
T4 ligation buffer   2uL
Nuclease fee water   4.5uL
Total volume   20uL


 
 
 
LB Agar Plate Prep
    • 0.9 g agar-agar power and 1.25g LB Broth and made it up to 50ml
    • Autoclaved at 121 degree for 15min
    • After autoclaving, aga was cooled to between 45°C and 50°C before pouring the plates to minimize the amount of condensation that forms
    • Added 50ul of 100mg/ml amp to the LB Agar before plating
    • 2 plates made and stored in 4 degree fridge
 

Friday - 9/8/2023

Freshly prepared LB- growth media
 
    • 0.25g LB broth in 10ml distilled H2O
    • Autoclaved at 121 degree for 15 min
Transformation
    • DH5 α comp cells obtained from -80 freezer and kept in ice for 15- 20 min
    • Keep an empty 2ml tube on ice
    • Take 100ul of comp cells into 2ml tubes, on ice
    • Add 5ul of plasmid DNA to comp cells. Don't mix. Flick it one time and tap on the counter 3-4 times
    • Incubate in ice for 30min.
    • Heat shock at 42 degree for 100sec and incubate in ice for 10 min.
    • Do no mix the sample at any of the above steps.
    • Add 800 ul LB into this mixture
    • Incubate in shaker at 37 degree 220 rpm for 1:30 hr
    • Pipette out 700 ul supernatent
    • Resuspend the cells in remaining 200ul and plate the entire sample
    • Plates kept for overnight incubation at 37 degree
( to be checked at 8:30 pm)
 
Plating
    • Plated 100 ul of comp cells and 200 ul of sample
    • Comp cells labelled - iGEM'23 gsα (1) negative control 8/9/23
    • IGEM'23 gsα (1) transformants 8/9/23

Saturday - 9/2/2023

  • Preparation of samples- FRET aptamerCort truncated sensitivity experiment
  • Different concentration of cortisol -:
10ng/ml - 5μl of 200ng/ml cort in 95μl Mq
50ng/ml- 25μl of 200ng/ml cort in 75μl Mq
100ng/ml- 50μl of 200ng/ml cort in 50μl Mq
150ng/ml-75μl of 200ng/ml cort in 15μl Mq
200ng/ml- take 100μl
250ng/ml-12.5μl of 0.02mg/ml cort in 87.5μl Mq
300ng/ml-15μl of 0.02mg/ml cort in 85μl Mq
350ng/ml-17.5μl of 0.02mg/ml cort in 82.5μl Mq
400ng/ml-20μl of 0.02mg/ml cort in 80μl Mq
  • FRET aptamertruncated cortisol - 0.5μM 600μl- take 30μl of 10μM sample in 570μl Mq
  • CDNA truncated cortisol- 1μM 600μl - take 60μl of 10μM sample in 540μl Mq
  • We had three controls- Mq, only FRET aptamer, only cDNA
  • We wanted the final volume of each well to be 150μl- so we took 50μl of FRET aptamer +50μl cDNA + 50μl cortisol of specific concentration
  • Order of samples in plate with the well number:
MQ FRET aptamer cDNA FRET aptamer +cDNA + 0ng/ml cortisol FRET aptamer +cDNA + 10ng/ml cortisol FRET aptamer +cDNA + 50 ng/ml cortisol FRET aptamer +cDNA + 100ng/ml cortisol FRET aptamer +cDNA + 150 ng/ml cortisol FRET aptamer +cDNA + 200ng/ml cortisol FRET aptamer +cDNA + 250 ng/ml cortisol FRET aptamer +cDNA + 300ng/ml cortisol FRET aptamer +cDNA + 350ng/ml cortisol FRET aptamer +cDNA + 400ng/ml cortisol
A1 B1 C1 H1 D1 E1 F1 G1 A2 B2 C2 D2 E2
  • Plate reader protocol for sensitivity experiments-
Take a 96 well plate (We can add a maximum of  200μl of sample in each well of the plate). We used the Greiner 96 Flat Bottom Transparent Polystyrene Cat. No.: 655101/655161/655192 [GRE96ft.pdfx] for our experiments.
Add 100μl MilliQ as control in the first well.
50μl APTAMER + 50μl MilliQ was added and pipette mixed in the next well as second control.
50μl CDNA + 50μl MilliQ was added and pipette mixed in the next well as third control.
Add 50μl APTAMER + 50μl cDNA in the next well and pipette mix it. ( molar ratio of aptamer: cDNA is kept constant )
Turn on the Tecan plate reader. [Infinite M plex TECAN plate reader].
Keep the plate in the plate reader. Set the protocol to read fluorescence with excitation at 490 nm and emission at 520 nm. We took 4 readings per well at every 10 minute interval for 90 minutes.
After 90 min, take out the plate and add different concentrations of cortisol, from 0 to 400ng/ml, in consecutive wells.
Make all the wells upto 150μl.
Again take fluoroscence readings with excitation at 490 nm and emission at 520 nm. We took 4 readings per well at every 10 minute interval for 60 minutes.
After 60 min, take out the plate.
Analyze the collected data and plot the graph.

Monday - 9/4/2023

  • Aptasensor formation experiment for FRETserotonin aptamer and cDNA 1 (all concentrations) - to check the optimal time required for FRET aptamer serotonin with cDNA 1 to bind at specific concentrations.
    • Preparation of sample-
      • For FRET aptamer0.25 micro molar , add 12.5 microlitre of 10 micro molar stock in 487.5 microlitre milliQ to get 500 microlitre sample.
      • For different concentrations of cdna 1
0.25μM 0.50μM 0.75μM 1μM 1.25μM Molar concentration
1.875 3.75 5.625 7.5 9.375 10 micromolar cDNA sample
73.175 71.25 69.375 67.5 65.625 Milli Q
Order of sample- It was added in the 5th column.
MQ FRET aptamer serotonin 1:1 1:2 1:3 1:4 1:5
 
  • Plate reader protocol for aptasensor formation experiment-
Take a 96 well plate (We can add a maximum of  200μl of sample in each well of the plate). We used the Greiner 96 Flat Bottom Transparent Polystyrene Cat. No.: 655101/655161/655192 [GRE96ft.pdfx] for our experiments.
Add 200μl MilliQ as control in the first well.
100μl APTAMER + 100μl MilliQ was added and pipette mixed in the next well as second control.
Add 100μl APTAMER + 100μl cDNA in the next well and pipette mix it.
Keep increasing concentrations of cDNA in each well. We used concentrations from 1:1 to 1:5 of aptamer:cDNA.
Turn on the Tecan plate reader. [Infinite M plex TECAN plate reader].
Keep the plate in the plate reader Set the protocol to read fluorescence with excitation at 490 nm and emission at 520 nm. We took 4 readings per well at every 10 minute interval for 90 minutes.
After 90 min, take out the plate.
Analyze the collected data and plot the graph.

Tuesday - 9/5/2023

  • Aptasensor formation experiment for FRETserotonin aptamer and cDNA 2 (all concentrations) - to check the optimal time required for FRET aptamer serotonin with cDNA 2 to bind at specific concentrations.
    • Preparation of sample-
      • For FRET aptamer0.25 micro molar , add 25 microlitre of 10 micro molar stock in 975 microlitre milliQ to get 1000 microlitre sample.
      • For different concentrations of cdna 2
0.25μM 0.50μM 0.75μM 1μM 1.25μM Molar concentration
3.75 7.5 11.25 15 18.75 10 micromolar cDNA sample
146.25 142.5 138.75 135 131.25 Milli Q
Order of sample- It was added in the 6th and 7th column replicates.
MQ FRET aptamer serotonin 1:1 1:2 1:3 1:4 1:5
 
  • Plate reader protocol for aptasensor formation experiment-
Take a 96 well plate (We can add a maximum of  200μl of sample in each well of the plate). We used the Greiner 96 Flat Bottom Transparent Polystyrene Cat. No.: 655101/655161/655192 [GRE96ft.pdfx] for our experiments.
Add 200μl MilliQ as control in the first well.
100μl APTAMER + 100μl MilliQ was added and pipette mixed in the next well as second control.
Add 100μl APTAMER + 100μl cDNA in the next well and pipette mix it.
Keep increasing concentrations of cDNA in each well. We used concentrations from 1:1 to 1:5 of aptamer:cDNA.
Turn on the Tecan plate reader. [Infinite M plex TECAN plate reader].
Keep the plate in the plate reader Set the protocol to read fluorescence with excitation at 490 nm and emission at 520 nm. We took 4 readings per well at every 10 minute interval for 90 minutes.
After 90 min, take out the plate.
Analyze the collected data and plot the graph.
 
  • Aptasensor formation experiment for FRET cortisol aptamer and cDNA 1 and cDNA 2 (all concentrations) - to check the optimal time required for FRET aptamer cortisol with cDNA to bind at specific concentrations.
      • Preparation of sample-
        • For FRET aptamer0.25 micro molar , add 35 microlitre of 10 micro molar stock in 1365 microlitre milliQ to get 1400 microlitre sample.
        • For different concentrations of cdna 2 (150microlitre) with replicate -
0.25μM 0.50μM 0.75μM 1μM 1.25μM Molar concentration
3.75 7.5 11.25 15 18.75 10 micromolar cDNA sample
146.25 142.5 138.75 135 131.25 Milli Q
        • For different concentrations of cdna 1 (75microlitre) -
 
0.25μM 0.50μM 0.75μM 1μM 1.25μM Molar concentration
1.875 3.75 5.625 7.5 9.375 10 micromolar cDNA sample
73.175 71.25 69.375 67.5 65.625 Milli Q
Order of sample- It was added in the 1st (FRET cort+cDNA 1 ), 3rd(FRET cort +cDNA 2) and 5th (FRET cort+ cDNA 2 ) column.
MQ FRET aptamer serotonin 1:1 1:2 1:3 1:4 1:5
 
    • Plate reader protocol for aptasensor formation experiment-
Take a 96 well plate (We can add a maximum of  200μl of sample in each well of the plate). We used the Greiner 96 Flat Bottom Transparent Polystyrene Cat. No.: 655101/655161/655192 [GRE96ft.pdfx] for our experiments.
Add 200μl MilliQ as control in the first well.
100μl APTAMER + 100μl MilliQ was added and pipette mixed in the next well as second control.
Add 100μl APTAMER + 100μl cDNA in the next well and pipette mix it.
Keep increasing concentrations of cDNA in each well. We used concentrations from 1:1 to 1:5 of aptamer:cDNA.
Turn on the Tecan plate reader. [Infinite M plex TECAN plate reader].
Keep the plate in the plate reader Set the protocol to read fluorescence with excitation at 490 nm and emission at 520 nm. We took 4 readings per well at every 10 minute interval for 90 minutes.
After 90 min, take out the plate.
Analyze the collected data and plot the graph.

Wednesday - 9/6/2023

  • Aptasensor formation experiment for FRET cortisol aptamer and cDNA 1 (all concentrations) with replicates - to check the optimal time required for FRET aptamer cortisol with cDNA 1 to bind at specific concentrations.
    • Preparation of sample-
      • For FRET aptamer0.25 micro molar , add 23.75 microlitre of 10 micro molar stock in 926.25 microlitre milliQ to get 950 microlitre sample.
      • For different concentrations of cdna 2
0.25μM 0.50μM 0.75μM 1μM 1.25μM Molar concentration
3.75 7.5 11.25 15 18.75 10 micromolar cDNA sample
146.25 142.5 138.75 135 131.25 Milli Q
Order of sample- It was added in the 10th and 12th column (replicates).
MQ FRET aptamer serotonin 1:1 1:2 1:3 1:4 1:5
 
  • Plate reader protocol for aptasensor formation experiment-
Take a 96 well plate (We can add a maximum of  200μl of sample in each well of the plate). We used the Greiner 96 Flat Bottom Transparent Polystyrene Cat. No.: 655101/655161/655192 [GRE96ft.pdfx] for our experiments.
Add 200μl MilliQ as control in the first well.
100μl APTAMER + 100μl MilliQ was added and pipette mixed in the next well as second control.
Add 100μl APTAMER + 100μl cDNA in the next well and pipette mix it.
Keep increasing concentrations of cDNA in each well. We used concentrations from 1:1 to 1:5 of aptamer:cDNA.
Turn on the Tecan plate reader. [Infinite M plex TECAN plate reader].
Keep the plate in the plate reader Set the protocol to read fluorescence with excitation at 490 nm and emission at 520 nm. We took 4 readings per well at every 10 minute interval for 90 minutes.
After 90 min, take out the plate.
Analyze the collected data and plot the graph.

Friday - 9/8/2023

  • Aptasensor formation experiment for FRET truncated cortisol aptamer and cDNA 1 truncated (all concentrations), and serotonin aptamer with cDNA1 with replicates - to check the optimal time required for FRET aptamer with cDNA to bind at specific concentrations.
    • Preparation of sample-
      • For FRET aptamertrn 0.5 micro molar , add 47.5 microlitre of 10 micro molar stock in 902.5microlitre milliQ to get 950 microlitre sample.
      • For FRET aptamerser 0.25 micro molar , add 12.5 microlitre of 10 micro molar stock in 487.5microlitre milliQ to get 500 microlitre sample.
      • For different concentrations of cdna 1 ser-
0.25μM 0.50μM 0.75μM 1μM 1.25μM Molar concentration
1.875 3.75 5.625 7.5 9.375 10 micromolar cDNA sample
73.175 71.25 69.375 67.5 65.625 Milli Q
      • For different concentrations of CDNA truncated- (with replicate)
0.25μM 0.50μM 0.75μM 1μM 1.25μM Molar concentration
3.75 7.5 11.25 15 18.75 10 micromolar cDNA sample
146.25 142.5 138.75 135 131.25 Milli Q
Order of sample- It was added in the 1st and 3rd column ( FRET aptamer trn cDNA trn replicates), 5th column (FRET aptamer serotonin cDNA 1)
MQ FRET aptamer serotonin 1:1 1:2 1:3 1:4 1:5
 
  • Plate reader protocol for aptasensor formation experiment-
Take a 96 well plate (We can add a maximum of  200μl of sample in each well of the plate). We used the Greiner 96 Flat Bottom Transparent Polystyrene Cat. No.: 655101/655161/655192 [GRE96ft.pdfx] for our experiments.
Add 200μl MilliQ as control in the first well.
100μl APTAMER + 100μl MilliQ was added and pipette mixed in the next well as second control.
Add 100μl APTAMER + 100μl cDNA in the next well and pipette mix it.
Keep increasing concentrations of cDNA in each well. We used concentrations from 1:1 to 1:5 of aptamer:cDNA.
Turn on the Tecan plate reader. [Infinite M plex TECAN plate reader].
Keep the plate in the plate reader Set the protocol to read fluorescence with excitation at 490 nm and emission at 520 nm. We took 4 readings per well at every 10 minute interval for 90 minutes.
After 90 min, take out the plate.
Analyze the collected data and plot the graph.

Sunday - 10/9/2023

Making 50mg /ml particles- 

add 0.5g particles to 10 ml solution

 To make TE buffer with 5mM mg2+ conc (using the TE buffer used for resuspension).

0.1 g MgCl2 into 100 ml TE (pH-8 )

 Make 132a and b and 124 a and b

To get a 500 µL 200 nM solution, mix 10 µL of 10 µM oligo stock with 490 µL of water or TE buffer.

 for C To get a 1000 µL 600 nM solution, mix 60 µL of 10 µM oligo stock with 940 µL of water or TE buffer.

make c working stock

To get a 200 µL 10 µM solution, mix 20 µL of 100 µM oligo stock with 180 µL of water or TE buffer.

 

Tuesday - 12/9/2023

To get a 100 µL 0.5 µM solution of A,B, mix 5 µL of 10 µM oligo stock with 95 µL of water or TE buffer.

 attachment of sequence b to particle

  • 1 ml 50 mg magnetic nanoparticle + 1 ml MES wash (this step avoids agglomeration)
  • absorb using magnet
  • remove supernatant
  • repeat wash
  • absorb and remove supernatant
  • add 100 ul mes and 100 ul EDS - blend for 30 min at room temperature
  • remove supernatant
  • add 200 ul dilute b (200nm conc)
  • keep in mixer overnight at room temperature
  • remove supernatant
  • add 200 ul dilute a (200nm conc)
  • add 200 ul dilute c (600nm conc)
  • incubate for 1 hr
  • remove supernatant
  • add 50 ul TE buffer
  • wash with it once, remove the supernatant
  • add 50 ul TE buffer and it's done

Saturday - 9/9/2023

LB AGAR PLATE PREP
prepared and poured 10 new LB Agar plates
 
TBE BUFFER STOCK PREPARED
500 mL from 50X TBE
 
Sunday - 9/10/2023
2 colonies observed after 24 h incubation
 


MASTERPLATE PREPARATION
  • Switched on the LAF and cleaned the working space with 70% Ethanol after sterilization.
  • A colony on the transformation plate is gently touched with an autoclaved pipette tip and immediately
placed in contact with the surface of the new masterplate and spread over a small area. Even pressure is applied to the tip.
  • The pipette tip is discarded into the pre prepared PCR tubes in order to run a colony PCR
  • Master plates are labeled with date and colonies 1 & 2
COLONY PCR
  • -Three mastermix tubes prepared and labelled - Control, C1 , C2
  • Pipette tubes after plating colony 1 and 2 discarded into respective tubes. Control didn't have any pipette tubes.
 
  con Control C1 C2
NEB Buffer   2.5 uL 2.5 uL 2.5 uL
dNTPSs 200uM 1.5uL 1.5uL 1.5uL
Forward primer 0.2uM 0.5 uL 0.5 uL 0.5 uL
Reverse primer 0.2uM 0.5 uL 0.5 uL 0.5 uL
Origin Taq polymerase   0.5 uL 0.5 uL 0.5 uL
Nuclease free water   19.5 uL 19.5 uL 19.5 uL
Total volume   25 uL 25 uL 25 uL
Thermocycler protocols saved under IGEMGSAP
 
AGAR GEL ELECTROPHORESIS
  • Made 60 mL1x TBE buffer - 6mL TBE + 54 mL milliQ
  • 0.6g low EEO agarose
  • Melt for 1 min 30 secs, streaked with 2 uL EtBr, left to solidify for 45 mins
  • Loaded 25uL sample + 5 uL loading dye
  • 2.5 uL NEB ladder + 2 uL purple loading dye
  • Ran gel at 90 v, 375 mA for 1 h 30 mins
No bands observed -TRANSFORMATION 1.0 didn't work.
INSERT CHEMIDOC IMAGE
10 sep colony pcr.scn
 
COLONY PCR 2.0

Monday - 9/11/2023

 
 
COLONY PCR 2.0
Did colony pcr again along with positive controls to check PCR conditions
    • cp 1- colony 1
    • cp2- colony 2
    • P(T)- Positive control from thejus
    • P(G)-Positive Control gsα
 
    CP1 CP2 P(G) P(T)
NEB Buffer   2.5 uL 2.5 uL 2.5 uL  
dNTPSs 200uM 1.5 uL 1.5 uL 1.5 uL 1.5 uL
Forward primer 0.2uM 0.5 uL 0.5 uL 0.5 uL 0.5 uL
Reverse primer 0.2uM 0.5 uL 0.5 uL 0.5 uL 0.5 uL
Origin Taq polymerase   0.5 uL 0.5 uL 0.5 uL 0.5 uL
Template   - - 0.2 uL 0.5uL
Nuclease free water   19.5 uL 19.5 uL 19.5 uL 19.5 uL
Total volume   25 uL 25 uL 25 uL 25 uL
 
Contamination was observed in prepared masterplates after 24 h - might be owing to improper working conditions in the LAF- faulty airflow
 



 
 
Tuesday - 9/12/2023
 
TRANSFORMATION 1.1
      • Repeated transformation 1.0 procedure
      • Used Comp cells from Tejas instead
      • negative control=- 100ul of comp cells and transformants
Wednesday - 9/13/2023
 
Did gsα PCR AMPLIFICATION
  • Prepared 5 tubes
Ran AGAROSE GEL ELECTRPHORESIS
 


ELUTION- 3 tubes
gsα 1 - 5.6ng/uL
gsα 2- 5.86 ng/uL
gsα 3 - 7.14 ng/uL
 
RESTRICTION DIGESTION
  • Prepared two tubes - one each containing insert and vector respectively. both are cut using the same restriction enzymes which are nde1 and bamh1
  • For gsα - tube 3
  • For vector - sandhya lab
  • For restriction digestion and cutsmart buffer - natesh lab
 
  INSERT gsα TUBE 3 VECTOR PET 19B
To be cut 19 uL - 135 ng add around 150 ng of whatever con of vector they give and make total reaction upto 25uL
Cutsmart buffer 2 uL 2 uL
BamH1-HF 0.2 uL 0.2 uL
Nde1 HF 0.3 uL 0.3 uL
Nuclease free water 3.5uL  
Total volume 25uL 25uL
 
Labelled the dry bath with sticky note, asking them not to turn off the machine.

Thursday - 9/14/2023

Restriction digestion finished
Ran AGAROSE GEL ELECTROPHORESIS 1.0 gel - got bands
ELUTION
  • 166 mg insert, added400 uL NT1
  • 186mg vector, added 400 uL NT1


NANODROP
  • Insert - 6.48 ng/uL
  • Vector- 12.17 ng/uL
Checked transformation plate after 24 hours - no colonies
 



Friday - 9/15/2023
 
LIGATION
reaction set up at 4:45 am
Ligation finishes at 9 PM
  L2 Positive control P
Insert 8uL (50ng) -
Vector 4.5uL (53ng) 4,5uL
T4 buffer 2 uL 2uL
T4 Ligase 1uL 1uL
Nucelase free water 4.5uL 12.5uL
Total volume 20uL 20uL
 
 
LIGATION PCR
  L2a L2b INSERT Vector
Ligation mixture ( 1:3 vector:insert ratio - 1uL 1uL 0.5uL 0.2uL
FP (4uM) vector FP-- 1.25uL insert FP - 1.25uL 1.25 uL 1.25 uL
RP (4uM) insert RP - 1.25uL vector RP-1.25uL 1.25 uL 1.25 uL
Taq polymerase 0.5uL 0.5uL 0.5uL 0.5uL
Taq pol buffer 2.5uL 2.5uL 2.5uL 2.5uL
dNTPS 1uL 1uL 1uL 1uL
Nuclease free water 17.5 17.5 18 18.3
Total volume 25uL 25uL 25uL 25uL
  • Got RF and FP from Sandhya ma'am lab ( Keerthana Chechi provided)
  • RG 3 is forwards primer and RG 4 is reverse primer ( concentration 4uM )
 
TRANSFORMATION 2.0
 
Since transformation 1.1 didn't work, We changed the volume and plasmid dna from 5 to 2 ul and used comp cells from Tejas lab
 

Sunday - 9/10/2023

  • Preparation of samples- sensitivity experiment- FRET aptamer serotonin and cDNA 1 and cDNA 2, FRET aptamer cortisol cDNA1 and FRET truncated aptamer cortisol cDNA truncated
  • Different concentration of cortisol -: (*5)
10ng/ml - 5μl of 200ng/ml cort in 95μl Mq
50ng/ml- 25μl of 200ng/ml cort in 75μl Mq
100ng/ml- 50μl of 200ng/ml cort in 50μl Mq
150ng/ml-75μl of 200ng/ml cort in 15μl Mq
200ng/ml- take 100μl
250ng/ml-12.5μl of 0.02mg/ml cort in 87.5μl Mq
300ng/ml-15μl of 0.02mg/ml cort in 85μl Mq
350ng/ml-17.5μl of 0.02mg/ml cort in 82.5μl Mq
400ng/ml-20μl of 0.02mg/ml cort in 80μl Mq
  • Different concentration of serotonin -: (*5)
10ng/ml - 5μl of 200ng/ml cort in 95μl Mq
50ng/ml- 25μl of 200ng/ml cort in 75μl Mq
100ng/ml- 50μl of 200ng/ml cort in 50μl Mq
150ng/ml-75μl of 200ng/ml cort in 15μl Mq
200ng/ml- take 100μl
250ng/ml-12.5μl of 0.02mg/ml cort in 87.5μl Mq
300ng/ml-15μl of 0.02mg/ml cort in 85μl Mq
350ng/ml-17.5μl of 0.02mg/ml cort in 82.5μl Mq
400ng/ml-20μl of 0.02mg/ml cort in 80μl Mq
  • FRET aptamertruncated cortisol - 0.5μM 1700μl- take 85μl of 10μM sample in 1615μl Mq
  • FRET aptamer cortisol - 0.25μM 1700μl- take 42.5μl of 10μM sample in 1657.5μl Mq
  • FRET aptamer serotonin- 0.25μM 3400μl- take 85μl of 10μM sample in 3315μl Mq
  • CDNA truncated cortisol- 1μM 1550μl - take 116.25μl of 10μM sample in 1433.75μl Mq
  • CDNA1 cortisol- 0.75μM 1550μl - take 155μl of 10μM sample in 1345μl Mq
  • CDNA1 serotonin - 0.75μM 1600μl - take 120μl of 10μM sample in 1480μl Mq
  • We had three controls- Mq, only FRET aptamer, only cDNA
  • We wanted the final volume of each well to be 150μl- so we took 50μl of FRET aptamer +50μl cDNA + 50μl cortisol of specific concentration
  • Order of samples in plate with the well number: ROW A and C ( FRET Ser and cDNA 1), ROW E and G(FRET Ser and cDNA 2), ROW B and D ( FRET Cort and cDNA1), ROW F and H ( FRET truncated cortisol and cDNA truncated)
MQ FRET aptamer cDNA FRET aptamer +cDNA + 0ng/ml cortisol FRET aptamer +cDNA + 10ng/ml cortisol FRET aptamer +cDNA + 50 ng/ml cortisol FRET aptamer +cDNA + 100ng/ml cortisol FRET aptamer +cDNA + 150 ng/ml cortisol FRET aptamer +cDNA + 200ng/ml cortisol FRET aptamer +cDNA + 250 ng/ml cortisol FRET aptamer +cDNA + 300ng/ml cortisol FRET aptamer +cDNA + 350ng/ml cortisol FRET aptamer +cDNA + 400ng/ml cortisol
A1 B1 C1 H1 D1 E1 F1 G1 A2 B2 C2 D2 E2
  • Plate reader protocol for sensitivity experiments-
Take a 96 well plate (We can add a maximum of  200μl of sample in each well of the plate). We used the Greiner 96 Flat Bottom Transparent Polystyrene Cat. No.: 655101/655161/655192 [GRE96ft.pdfx] for our experiments.
Add 100μl MilliQ as control in the first well.
50μl APTAMER + 50μl MilliQ was added and pipette mixed in the next well as second control.
50μl CDNA + 50μl MilliQ was added and pipette mixed in the next well as third control.
Add 50μl APTAMER + 50μl cDNA in the next well and pipette mix it. ( molar ratio of aptamer: cDNA is kept constant )
Turn on the Tecan plate reader. [Infinite M plex TECAN plate reader].
Keep the plate in the plate reader. Set the protocol to read fluorescence with excitation at 490 nm and emission at 520 nm. We took 4 readings per well at every 10 minute interval for 90 minutes.
After 90 min, take out the plate and add different concentrations of cortisol, from 0 to 400ng/ml, in consecutive wells.
Make all the wells upto 150μl.
Again take fluoroscence readings with excitation at 490 nm and emission at 520 nm. We took 4 readings per well at every 10 minute interval for 60 minutes.
After 60 min, take out the plate.
Analyze the collected data and plot the graph.
 
  • Incubation Time experiment for FRET aptamer cortisol and cDNA truncated (1:3 concentration) - to check the optimal time required for FRET aptamer cortisol with cDNA truncated to bind at specific concentrations.
  • Preparation of sample-
      • For FRET aptamer cortisol 0.25 micro molar , add 5 microlitre of 10 micro molar stock in 195 microlitre milliQ to get 200microlitre sample.
      • For cdna truncated cortisol 0.75 micro molar , add 7.5 microlitre of 10 micro molar stock in 92.5 microlitre milliQ to get 100microlitre sample.
  • Experimental setup-
1) Take readings with 200μl MilliQ
2) Take 100μl 0.25μM FRET aptamer cortisol and 100μl MilliQ. Pipette mix it and take reading.
3) Take 100μl 0.25μM FRET aptamer cortisol and 100μl 0.5μM cDNAtruncated cortisol. Pipette mix it and measure the change in fluorescence every 10 mins for 90 min.
  • Fluorolog Protocol:
          • We have a 200μl cuvette .
          • First, we need to wash the cuvette. Wash the cuvette thoroughly first with milliQ, then with acetone and finally with ethanol.
          • Now, wash the cuvette with autoclaved Milli-Q once before taking 200μl MilliQ in the cuvette to take the base reading.
          • Wipe the cuvette with tissue and place it in the spectrofluorometer.
          • Set the protocol on the computer. We took a range scan to get emission readings between 495nm -700nm when the excitation peak was set at 495nm.
          • Run the protocol.
          • Empty the cuvette and wash thoroughly with MilliQ.
          • Add 200μl of sample in the cuvette. Wipe with a tissue and run the same protocol on the computer.
          • After taking all readings, wash the cuvette with acetone, ethanol and MilliQ thoroughly.

Wednesday - 9/13/2023

Sensitivity of FRET aptamer Cortisol-
  • Preparation of samples- FRET aptamerCort sensitivity experiment
  • Different concentration of cortisol -:
10ng/ml - 5μl of 200ng/ml cort in 95μl Mq
50ng/ml- 25μl of 200ng/ml cort in 75μl Mq
100ng/ml- 50μl of 200ng/ml cort in 50μl Mq
150ng/ml-75μl of 200ng/ml cort in 15μl Mq
200ng/ml- take 100μl
250ng/ml-12.5μl of 0.02mg/ml cort in 87.5μl Mq
300ng/ml-15μl of 0.02mg/ml cort in 85μl Mq
350ng/ml-17.5μl of 0.02mg/ml cort in 82.5μl Mq
400ng/ml-20μl of 0.02mg/ml cort in 80μl Mq
  • FRET aptamer cortisol - 0.25μM 1700μl- take 4.25μl of 100μM sample in 1695.75μl Mq
  • CDNA1 cortisol- 0.75μM 1600μl - take 12μl of 100μM sample in 1588μl Mq
  • We had two controls- Mq, only FRET aptamer
  • We wanted the final volume of each well to be 150μl- so we took 50μl of FRET aptamer +50μl cDNA + 50μl cortisol of specific concentration
  • Order of samples in plate with the well number:
MQ FRET aptamer cDNA FRET aptamer +cDNA + 0ng/ml cortisol FRET aptamer +cDNA + 10ng/ml cortisol FRET aptamer +cDNA + 50 ng/ml cortisol FRET aptamer +cDNA + 100ng/ml cortisol FRET aptamer +cDNA + 150 ng/ml cortisol FRET aptamer +cDNA + 200ng/ml cortisol FRET aptamer +cDNA + 250 ng/ml cortisol FRET aptamer +cDNA + 300ng/ml cortisol FRET aptamer +cDNA + 350ng/ml cortisol FRET aptamer +cDNA + 400ng/ml cortisol
A1 B1 C1 D1 E1 F1 G1 H1 A2 B2 C2 D2 E2
  • Plate reader protocol for sensitivity experiments-
Take a 96 well plate (We can add a maximum of  200μl of sample in each well of the plate). We used the Greiner 96 Flat Bottom Transparent Polystyrene Cat. No.: 655101/655161/655192 [GRE96ft.pdfx] for our experiments.
Add 100μl MilliQ as control in the first well.
50μl APTAMER + 50μl MilliQ was added and pipette mixed in the next well as second control.
50μl CDNA + 50μl MilliQ was added and pipette mixed in the next well as third control.
Add 50μl APTAMER + 50μl cDNA in the next well and pipette mix it. ( molar ratio of aptamer: cDNA is kept constant )
Turn on the Tecan plate reader. [Infinite M plex TECAN plate reader].
Keep the plate in the plate reader. Set the protocol to read fluorescence with excitation at 490 nm and emission at 520 nm. We took 4 readings per well at every 10 minute interval for 90 minutes.
After 90 min, take out the plate and add different concentrations of cortisol, from 0 to 400ng/ml, in consecutive wells.
Make all the wells upto 150μl.
Again take fluoroscence readings with excitation at 490 nm and emission at 520 nm. We took 4 readings per well at every 10 minute interval for 60 minutes.
After 60 min, take out the plate.
Analyze the collected data and plot the graph.

Thursday - 9/14/2023

Biomarker mix experiment protocol-
  • Plate reader protocol for biomarker mix experiments-
  • Take a 96 well plate (We can add a maximum of 200μl of sample in each well of the plate). We used the Greiner 96 Flat Bottom Transparent Polystyrene Cat. No.: 655101/655161/655192 [GRE96ft.pdfx] for our experiments.
  • Add 150μl MilliQ as control in the first well.
  • Add 75μl APTAMER + 75μl cDNA in the next six wells and pipette mix it. ( molar ratio of aptamer: cDNA is kept constant )
  • Turn on the Tecan plate reader. [Infinite M plex TECAN plate reader].
  • Keep the plate in the plate reader. Set the protocol to read fluorescence with excitation at 490 nm and emission at 520 nm. We took 4 readings per well at every 10 minute interval for 90 minutes (for formation of the quantifier)
  • After 90 min, take out the plate and add different biomarkers ( 50μl - In order )
 
MQ Quantifier Quantifier+ Serotonin Quantifier+ Cortisol Quantifier+ miDNA 132 Quantifier+ miDNA 124 Mix
  • Mix- 12.5μl of each biomarker.
  • Make the final volume of all the wells 200μl.
  • Again take fluorescence readings with excitation at 490 nm and emission at 520 nm. We took 4 readings per well at every 10 minute interval for 60 minutes.
  • After 60 min, take out the plate.
  • Analyze the collected data and plot the graph.
Experimental setup-
  • ROW E and G for FRET aptamer serotonin and cDNA1 (with replicate)

Friday - 9/15/2023

Biomarker mix for FRET aptamerser and cDNA1 quantifier and FRET aptamertrn and cDNA trn quantifier
Experimental setup-
  • ROW Band D for FRET aptamer serotonin and cDNA1 (with replicate)
  • ROW F and H for FRET aptamer truncated cortisol and cDNA truncated (with replicate)
Biomarker mix experiment protocol-
  • Plate reader protocol for biomarker mix experiments-
  • Take a 96 well plate (We can add a maximum of 200μl of sample in each well of the plate). We used the Greiner 96 Flat Bottom Transparent Polystyrene Cat. No.: 655101/655161/655192 [GRE96ft.pdfx] for our experiments.
  • Add 150μl MilliQ as control in the first well.
  • Add 75μl APTAMER + 75μl cDNA in the next six wells and pipette mix it. ( molar ratio of aptamer: cDNA is kept constant )
  • Turn on the Tecan plate reader. [Infinite M plex TECAN plate reader].
  • Keep the plate in the plate reader. Set the protocol to read fluorescence with excitation at 490 nm and emission at 520 nm. We took 4 readings per well at every 10 minute interval for 90 minutes (for formation of the quantifier)
  • After 90 min, take out the plate and add different biomarkers ( 50μl - In order )
 
MQ Quantifier Quantifier+ Serotonin Quantifier+ Cortisol Quantifier+ miDNA 132 Quantifier+ miDNA 124 Mix
  • Mix- 12.5μl of each biomarker.
  • Make the final volume of all the wells 200μl.
  • Again take fluorescence readings with excitation at 490 nm and emission at 520 nm. We took 4 readings per well at every 10 minute interval for 60 minutes.
  • After 60 min, take out the plate.
  • Analyze the collected data and plot the graph.
 
Fluorolog FRET aptamer serotonin and cdna trn to check formation of aptasensor-
  • Incubation Time experiment for FRET aptamer serotonin and cDNA truncated (1:3 concentration) - to check the optimal time required for FRET aptamer serotonin with cDNA truncated to bind at specific concentrations.
  • Preparation of sample-
      • For FRET aptamer serotonin 0.25 micro molar , add 5 microlitre of 10 micro molar stock in 195 microlitre milliQ to get 200microlitre sample.
      • For cdna truncated cortisol 0.75 micro molar , add 7.5 microlitre of 10 micro molar stock in 92.5 microlitre milliQ to get 100microlitre sample.
  • Experimental setup-
1) Take readings with 200μl MilliQ
2) Take 100μl 0.25μM FRET aptamer serotonin and 100μl MilliQ. Pipette mix it and take reading.
3) Take 100μl 0.25μM FRET aptamer serotonin and 100μl 0.75μM cDNAtruncated cortisol. Pipette mix it and measure the change in fluorescence every 10 mins for 90 min.
  • Fluorolog Protocol:
          • We have a 200μl cuvette .
          • First, we need to wash the cuvette. Wash the cuvette thoroughly first with milliQ, then with acetone and finally with ethanol.
          • Now, wash the cuvette with autoclaved Milli-Q once before taking 200μl MilliQ in the cuvette to take the base reading.
          • Wipe the cuvette with tissue and place it in the spectrofluorometer.
          • Set the protocol on the computer. We took a range scan to get emission readings between 495nm -700nm when the excitation peak was set at 495nm.
          • Run the protocol.
          • Empty the cuvette and wash thoroughly with MilliQ.
          • Add 200μl of sample in the cuvette. Wipe with a tissue and run the same protocol on the computer.
          • After taking all readings, wash the cuvette with acetone, ethanol and MilliQ thoroughly.

Tuesday - 9/19/2023

Transformation plates observed after 24 h incubation
Colonies were observed in both plates
 



Exam Week :)

Monday- 2/10/2023

Plate reader

c3 - te (108 ul)

c4 132 - (100p + 8 te)

c5 132 + dna (100p + 8 dna)

c6 124 - (100p + 8 te)

c7 - 124+dna (100p + 8 dna)

 Excitation scan
- 490 emission
-excitation - 500 -650 nm range (1 nm increase)

 Building probe -

making more seq a and b of 200 nm

To get a 1000 µL 200 nM solution, mix 20 µL of 10 µM oligo stock with 980 µL TE buffer

 for 600 nm C

 To get a 1000 µL 600 nM solution, mix 60 µL of 10 µM oligo stock with 940 µL of TE buffer.

 

 Tuesday- 3/10/2023

  • 1 ml 1 mg/ml magnetic nanoparticle + 1 ml MES wash (this step avoids agglomeration)
  • centrifuge, 8000 rpm for 5 min
  • remove supernatant
  • repeat wash (use a magnet instead of a centrifuge here)
  • absorb and remove supernatant
  • add 100 ul mes and 100 ul EDS
  • -blend for 30 minutes at room temperature
  • remove supernatant
  • add 200 ul dilute b (200nm conc)
  • Keep in mixer overnight at room temperature.
  • remove supernatant
  • add 200 ul dilute a (200nm conc)
  • add 200 ul dilute c (600nm conc)
  • incubate for 1 hr
  • remove supernatant
  • add 50 ul TE buffer
  • was with it once, remove the supernatant
  • add 50 ul TE buffer and it's done

 To make different conc

 500, 100, 50, 10 nm, 1 nm, 0.5nm, 0.1 nm, 0.01 nm = 10 pm

 500 µL 0.5 µM solution, mix 25 µL of 10 µM oligo stock with 475 µL of water or TE buffer.

  • 500 µL 0.1 µM solution, mix 100 µL of 0.5 µM oligo stock with 400 µL of water or TE buffer
  • 250 ul from 0.1 um+ 250 ul te = 50 nm
  • 100 ul from 50nm and 400 ul te = 10 nm
  • 50 ul 10nm 450 ul te = 1 nm
  • 50 ul 1nm 450 ul te = 0.1 nm
  • 50 ul 0.1nm 450 ul te = 0.01 nm = 10 pm

 Plate loading

D3 - probe 132+ 75ul te, D4 - 500 nm , D5- 100 nm, D6 - 50nm, D7 - 10nm, D8- 1 nm, D9 - 0.1nm, D10 - 0.01 nm

E3 - probe 124 + 75 ul te, E4 - 500 nm , E5- 100 nm, E6 - 50nm, E7 - 10nm, E8- 1 nm, E9 - 0.1nm, E10 - 0.01 nm

in everything probe = 25 ul

midna = 75 ul

measurements to be taken at excitation - 490 nm

Emission - 500 - 600 nm (based on previous data)

Wednesday - 10/4/2023

ITC Experiment- [FRET aptamer serotonin 0.25μM + cDNA1 serotonin 1μM- 1:1 conc]
Sample preparation-
  • FRET Apt Ser- for 200μl of 0.25μM - add 5μl of 10μM sample in 195μl MQ
  • cDNA1 Ser- for 80μl of 1μM- add 8μl of 10μM sample in 72μl MQ
ITC Protocol-
  • Prepare the sample and ligand solutions.
      • The sample solution should contain the macromolecule whose binding affinity is being measured which in our case was the aptamer. The ligand solution should contain the molecule that is binding to the sample which for experiment was the cDNA. The concentrations of the sample and ligand solutions should be chosen carefully to ensure that the binding reaction is in the measurable range of the ITC instrument.
      • The concentration we used was 0.25 micromolar and prepared 200 microliters of the aptamer to be inserted into the sample well, and 1 micromolar cDNA 80 microliter in volume was prepared to be titrated into the sample well.
    • Load the sample and ligand solutions into the ITC instrument. The ITC instrument has two cells: a sample cell and a reference cell. The sample solution is loaded into the sample cell and the ligand solution is loaded into the reference cell.
    • Equilibrate the sample and ligand solutions. The ITC instrument will equilibrate the sample and ligand solutions to the same temperature. This is important to ensure that the heat changes measured during the binding reaction are due only to the binding event.
    • Titrate the ligand solution into the sample cell. The ITC instrument will titrate the ligand solution into the sample cell in a series of small injections. The heat released or absorbed during each injection will be measured.

 

Friday - 10/6/2023

ITC Experiment- [FRET aptamer serotonin 0.25μM + cDNA2 serotonin 2μM- 1:2 conc]
Sample preparation-
  • FRET Apt Ser- for 200μl of 0.25μM - add 5μl of 10μM sample in 195μl MQ
  • cDNA2 Ser- for 80μl of 2μM- add 16μl of 10μM sample in 64μl MQ
ITC Protocol-
  • Prepare the sample and ligand solutions.
      • The sample solution should contain the macromolecule whose binding affinity is being measured which in our case was the aptamer. The ligand solution should contain the molecule that is binding to the sample which for experiment was the cDNA. The concentrations of the sample and ligand solutions should be chosen carefully to ensure that the binding reaction is in the measurable range of the ITC instrument.
      • The concentration we used was 0.25 micromolar and prepared 200 microliters of the FRET aptamer ser to be inserted into the sample well, and 2 micromolar cDNA 2 80 microliter in volume was prepared to be titrated into the sample well.
    • Load the sample and ligand solutions into the ITC instrument. The ITC instrument has two cells: a sample cell and a reference cell. The sample solution is loaded into the sample cell and the ligand solution is loaded into the reference cell.
    • Equilibrate the sample and ligand solutions. The ITC instrument will equilibrate the sample and ligand solutions to the same temperature. This is important to ensure that the heat changes measured during the binding reaction are due only to the binding event.
    • Titrate the ligand solution into the sample cell. The ITC instrument will titrate the ligand solution into the sample cell in a series of small injections. The heat released or absorbed during each injection will be measured.
 
  • Aptasensor formation experiment for FRET aptamer cortisol and cDNA 1 and cDNA2 (all concentrations), with replicates - to check the optimal time required for FRET aptamer with cDNA to bind at specific concentrations.
    • Preparation of sample-
      • For FRET aptamercort 0.25 micro molar , add 6 microlitre of 100 micro molar stock in 2394microlitre TE buffer to get 2400microlitre sample.
      • For different concentrations of cdna 1 and cDNA2 -
0.25μM 0.50μM 0.75μM 1μM 1.25μM Molar concentration
2.5 5 7.5 10 12.5 10 micromolar cDNA sample
97.5 95 92.5 90 87.5 TE buffer
Order of sample- It was added in the 9th and 10thcolumn ( FRET aptamer cortisol and cDNA 1 with replicates), 11th and 12th column (FRET aptamer cortisol cDNA 2 with replicates)
MQ FRET aptamer serotonin 1:1 1:2 1:3 1:4 1:5
 
  • Plate reader protocol for aptasensor formation experiment-
Take a 96 well plate (We can add a maximum of  200μl of sample in each well of the plate). We used the Greiner 96 Flat Bottom Transparent Polystyrene Cat. No.: 655101/655161/655192 [GRE96ft.pdfx] for our experiments.
Add 200μl MilliQ as control in the first well.
100μl APTAMER + 100μl MilliQ was added and pipette mixed in the next well as second control.
Add 100μl APTAMER + 100μl cDNA in the next well and pipette mix it.
Keep increasing concentrations of cDNA in each well. We used concentrations from 1:1 to 1:5 of aptamer:cDNA.
Turn on the Tecan plate reader. [Infinite M plex TECAN plate reader].
Keep the plate in the plate reader Set the protocol to read fluorescence with excitation at 490 nm and emission at 520 nm. We took 4 readings per well at every 10 minute interval for 90 minutes.
After 90 min, take out the plate.
Analyze the collected data and plot the graph.

Sunday- 8/10/2023

  • 1 ml 1 mg/ml magnetic nanoparticle + 1 ml MES wash (this step avoids agglomeration)
  • centrifuge, 8000 rpm for 5 min
  • remove supernatant
  • repeat wash (use a magnet instead of a centrifuge here)
  • absorb and remove supernatant
  • add 100 ul mes and 100 ul EDS
  • -blend for 30 minutes at room temperature
  • remove supernatant
  • add 200 ul dilute b (200nm conc)
  • Keep in mixer overnight at room temperature.
  • remove supernatant
  • add 200 ul dilute a (200nm conc)
  • add 200 ul dilute c (600nm conc)
  • incubate for 1 hr
  • remove supernatant
  • add 50 ul TE buffer
  • was with it once, remove the supernatant
  • add 50 ul TE buffer and it's done

Take four eppendorf tubes and label them as:

  1. Probe 124 + TE Buffer (control)
  2. Probe 124 + miDNA 132(10nM)
  3. Probe 124 + miDNA 124(10nM)
  4. Probe 124 + miDNA 124-2 (10nM)

Similarly prepare four eppendorf tubes for probe 132

  • Add 25uL probe in each tube
  • Add 75uL of of TE, miDNA 132, miDNA 124, miDNA 124-2, into respective labelled tubes at the time of loading
  • Do the same for probes 132
  • Take 100ul of the samples and pour it into the wells for plate reading

Plate loading

F3 - 132P + TE; F4 - 132P + miDNA 124; F5 - 132P + miDNA 132; F6 - 132P + miDNA 132-2

G3- 124P + TE; G4 - 124P + miDNA 124; F5- 124P + miDNA 132; G6 - 124P + miDNA 124-2

Measurement to be taken at an excitation of 490nm

Emission at 500-600nm range

Saturday - 10/7/2023

ITC Experiment- [FRET aptamer serotonin 0.25μM + serotonin ]
Sample preparation-
  • FRET Apt Ser- for 200μl of 0.25μM - add 5μl of 10μM sample in 195μl MQ
ITC Protocol-
  • Prepare the sample and ligand solutions.
      • The sample solution should contain the macromolecule whose binding affinity is being measured which in our case was the aptamer. The ligand solution should contain the molecule that is binding to the sample which for experiment was the cDNA. The concentrations of the sample and ligand solutions should be chosen carefully to ensure that the binding reaction is in the measurable range of the ITC instrument.
      • The concentration we used was 0.25 micromolar and prepared 200 microliters of the aptamer to be inserted into the sample well, and 2000ng/ml of serotonin 80 microliter in volume was prepared to be titrated into the sample well.
    • Load the sample and ligand solutions into the ITC instrument. The ITC instrument has two cells: a sample cell and a reference cell. The sample solution is loaded into the sample cell and the ligand solution is loaded into the reference cell.
    • Equilibrate the sample and ligand solutions. The ITC instrument will equilibrate the sample and ligand solutions to the same temperature. This is important to ensure that the heat changes measured during the binding reaction are due only to the binding event.
    • Titrate the ligand solution into the sample cell. The ITC instrument will titrate the ligand solution into the sample cell in a series of small injections. The heat released or absorbed during each injection will be measured.

Sunday - 10/8/2023

ITC Experiment- [FRET aptamer cortisol 0.25μM + cDNA1 cortisol 2μM- 1:2 conc]
Sample preparation-
  • FRET Apt cort- for 200μl of 0.25μM - add 5μl of 10μM sample in 195μl MQ
  • cDNA2 cort- for 80μl of 2μM- add 16μl of 10μM sample in 64μl MQ
ITC Protocol-
  • Prepare the sample and ligand solutions.
      • The sample solution should contain the macromolecule whose binding affinity is being measured which in our case was the aptamer. The ligand solution should contain the molecule that is binding to the sample which for experiment was the cDNA. The concentrations of the sample and ligand solutions should be chosen carefully to ensure that the binding reaction is in the measurable range of the ITC instrument.
      • The concentration we used was 0.25 micromolar and prepared 200 microliters of the aptamer to be inserted into the sample well and 2 micromolar cDNA 80 microliter in volume was prepared to be titrated into the sample well.
    • Load the sample and ligand solutions into the ITC instrument. The ITC instrument has two cells: a sample cell and a reference cell. The sample solution is loaded into the sample cell and the ligand solution is loaded into the reference cell.
    • Equilibrate the sample and ligand solutions. The ITC instrument will equilibrate the sample and ligand solutions to the same temperature. This is important to ensure that the heat changes measured during the binding reaction are due only to the binding event.
    • Titrate the ligand solution into the sample cell. The ITC instrument will titrate the ligand solution into the sample cell in a series of small injections. The heat released or absorbed during each injection will be measured.

Tuesday - 10/10/2023

ITC Experiment- [FRET aptamer cortisol 0.25μM + cortisol 2000ng/ml]
Sample preparation-
  • FRET Apt cort- for 200μl of 0.25μM - add 5μl of 10μM sample in 195μl MQ
ITC Protocol-
  • Prepare the sample and ligand solutions.
      • The sample solution should contain the macromolecule whose binding affinity is being measured which in our case was the aptamer. The ligand solution should contain the molecule that is binding to the sample which for experiment was the cDNA. The concentrations of the sample and ligand solutions should be chosen carefully to ensure that the binding reaction is in the measurable range of the ITC instrument.
      • The concentration we used was 0.25 micromolar and prepared 200 microliters of the aptamer to be inserted into the sample well and 2000ng/ml 80 microliter in volume was prepared to be titrated into the sample well.
    • Load the sample and ligand solutions into the ITC instrument. The ITC instrument has two cells: a sample cell and a reference cell. The sample solution is loaded into the sample cell and the ligand solution is loaded into the reference cell.
    • Equilibrate the sample and ligand solutions. The ITC instrument will equilibrate the sample and ligand solutions to the same temperature. This is important to ensure that the heat changes measured during the binding reaction are due only to the binding event.
    • Titrate the ligand solution into the sample cell. The ITC instrument will titrate the ligand solution into the sample cell in a series of small injections. The heat released or absorbed during each injection will be measured.

Aptamers

Nanoprobes

Gsα

Interlab

Back To Top ↑