(1) Reagents:
Eastep® Super Total RNA Extraction Kit
(2) Steps:
- Load an appropriate number of flies into a test tube and anesthetize them with carbon dioxide using a carbon dioxide anesthesia system.
- ransfer 15 flies to a lysis tube, and put the lysis tube in a -20°C freezer for 20 minutes to ensure the flies are dead.
- Add 300μl lysis solution and 300μl dilution solution to the lysis tube and mix thoroughly using a homogenizer.
- Heat the lysis tube in a metal heater at 70°C for 3 minutes, and let it stand at room temperature for 3-5 minutes.
- Centrifuge at 16,000g for 5 minutes.
-
Carefully collect the supernatant liquid and add 0.5 times the volume of absolute ethanol. Mix the solution by pipetting 20-30 times until it turns into a light blue turbid solution
with white foam.
- Transfer the mixture to a spin column and collection tube in two portions, centrifuge at 16,000g for 1 minute. Discard the flow-through.
- Add 600μl RNA wash solution, centrifuge at 16,000g for 1 minute. Discard the flow-through.
-
Add 5μl 10× DNA enzyme I buffer, 5μl DNA enzyme I, and 40μl 40 non-nuclease water to the center of the adsorbent membrane in each centrifuge tube. Let it stand at room temperature for
15 minutes.
- Centrifuge at 16,000g for 1 minute, and discard the flow-through.
- Place the spin column back onto the collection tube, and centrifuge at 16,000g for 2 minutes to ensure drying.
- Transfer the spin column to an elution tube, add 50-200μl non-nuclease water to the center of the spin column membrane, and let it stand at room temperature for 2 minutes.
- Centrifuge at 16,000g for 1 minute, measure the concentration and purity of RNA using a UV-visible spectrophotometer, and finally store the RNA at -70°C.
(1) Reagents:
PrimeScript™ RT Master Mix
(2) Steps:
1)Reverse Transcription Reaction
a. Prepare the RT reaction mixture as follows (prepare on ice):
| Reagent |
Amount |
Final Concentration |
| 5× PrimeScript RT Master Mix (Perfect Real Time) |
2μl |
× |
| Total RNA |
* |
|
| RNase Free dH2O |
up to 10μl |
|
* The reaction system can be scaled up as needed. A maximum of 500ng of total RNA can be used in a 10μl reaction system.
b. Perform the reverse transcription reaction after gentle mixing, under the following conditions: 37°C: 15 minutes (reverse transcription reaction); 85°C: 5 seconds (inactivation of
reverse transcriptase); 4°C (storage).
2)Real Time PCR
a. Prepare the PCR reaction mixture as follows (prepare on ice):
| Reagent |
Amount |
Volume |
Final Concentration |
| TB Green Premix Ex Taq II (Tli RNaseH Plus) (2×) |
10μl |
25μl |
1× |
| PCR Forward Primer (10μM) |
0.8μl |
2μl |
0.4μM |
| PCR Reverse Primer (10μM) |
0.8μl |
2μl |
0.4μM |
| ROX Reference Dye or Dye II (50×) |
0.4μl |
1μl |
1× |
| RT reaction mixture (cDNA solution) |
2μl |
4μl |
|
| Sterile water |
6μl |
16μl |
|
| Total |
20μl |
50μl |
|
b. Perform Real Time PCR using a two-step PCR program:
Stage 1: Denaturation
Stage 2: PCR Amplification
- Reps: 40
- 95°C: 5 seconds
- 60°C: 30 seconds
3)Analysis of Experimental Results
After the reaction is complete, analyze the amplification curve and melting curve of Real Time PCR, and prepare a standard curve for PCR quantification, etc.
(1) PCR System
- 5μl 10× Buffer for KOD-Plus
- 5μl 2mM dNTPs
- 2μl MgSO4
- 2μl cDNA
- 1.5μl Forward Primer
- 1.5μl Reverse Primer
- 1μl KOD-Plus (1.0U/μl)
- 32μl PCR grade water
PCR Program
- Hid
- MTF-1
(1) Agarose Gel Electrophoresis Detection.
(2) Gel Extraction.
Carefully cut out the gel containing the desired band using a surgical knife and place it into a PVC tube (try to ensure that the volume of each gel fragment is roughly equal).
(3) Gel Recovery:
- Weigh the gel fragment containing the band (try to ensure that the weight of each gel fragment is roughly equal, approximately 200mg)
- Add Gel Solubilization Buffer (GSB) with a volume three times that of the gel to dissolve the gel
- Incubate in a 55°C water bath for 10 minutes, intermittently taking out and mixing every 3 minutes until the gel block is completely melted
- Add an equal volume of isopropanol to reduce the solubility of DNA and ensure the recovery yield
-
Wait for GSB to cool to room temperature, transfer it to a centrifuge tube, let it stand for 1 minute, centrifuge at 10,000×g for 1 minute to ensure DNA is adsorbed onto the collection
column, and discard the flow-through
- Add 650μl of Wash Buffer (WB), centrifuge at 10,000×g for 1 minute to wash away non-specific large molecules and discard the flow-through
- Centrifuge at 10,000×g for 1 minute to remove residual WB at the bottom of the centrifuge tube
- Open the lid and let it stand for 1 minute to allow residual ethanol to evaporate
- Add 30μl of double-distilled water, and let it stand for 1 minute
- Centrifuge at 10,000×g for 1 minute to elute the DNA and obtain the DNA solution
- Store the DNA solution at -20°C
Determination of Concentration
- Transfer a drop of double-distilled water to the detection instrument for blank detection
-
Using a micropipette with the lowest range, and transfer a small drop of the DNA solution to the detection instrument (wipe the detection head with a sterile paper after each
measurement)
-
To test whether the machine is working properly and the range is reasonable, after the detection, use double-distilled water (with a concentration close to 0) and a prepared plasmid
solution (with a very high concentration) for testing
(1) Digestion
- Add 5μl Buffer solution, 25μl MTF, 1μl bgl II, 1μl XhoI, and 18μl H2O.
- Use water bath heating to keep the solution at 37°C for 3 to 5 hours
(2) Purification of Digested DNA
- Add Solution I (Binding Buffer; in the same volume as the digested DNA solution) and mix the solution by pipetting up and down
- Add the mixed sample to the DNA purification spin column and let it stand at room temperature for 1 minute
- Centrifuge the sample at 16000×g for 1 minute and discard the effluent that has flowed through the silica membrane
- Add 700μl of Solution II (Wash Buffer) to the spin purification column and let it stand at room temperature for 1 minute
- Centrifuge the sample at 16000×g for 1 minute and discard the effluent
- Add 500μl of Solution II again to the purification column and repeat step 5
-
After the effluent is discarded again, centrifuge the sample at 16000×g for 1 minute without adding any solution to the silica membrane, in order to dry it off of any buffer and allow
the ethanol-containing components to evaporate
- Transfer the DNA spin purification column into a collection tube, add 30μl ddH2O (pH>6.5), and let it stand at room temperature for 1 minute
- Centrifuge the sample at 16000×g for 1 minute to elute the DNA
- Determine the concentration of the DNA solution (effluent)
-
Set up the following reaction in a microcentrifuge tube on ice
| Component |
20μI Reaction |
| T4 DNA Ligase Buffer (10×)* |
2μI |
| Vector DNA (4kb) |
50ng (0.020pmol) |
| Insert DNA (1kb) |
37.5ng (0.060pmol) |
| Nuclease-free water |
to 20μI |
| T4 DNA Ligase |
1μI |
- Gently mix the reaction by pipetting up and down and microfuge briefly
- For cohesive (sticky) ends, incubate at 16℃ overnight or room temperature for 10 minutes
-
For blunt ends or single base overhangs, incubate at 16℃ overnight or room temperature for 2 hours (alternatively, high concentration T4 DNA Ligase can be used in a 10 minute ligation)
- Heat inactivate at 65℃ for 10 minutes
- Chill on ice and transfer 1-5μI of the reaction into 50μI competent cells
-
Take 100μl of competent cells that were thawed on ice, add target DNA (plasmid or Ligated DNA) into the suspension, mix the sample gently (through pipetting up and down or flicking
the walls of the tube several time), and let it stand on ice for 30 minutes.
Note: The target plasmid or ligand product should not be more than 1/10 of the total volume of the competent cell suspension.
-
Use a 42°C water heat bath to heat shock the competent cells for 45 to 60 seconds, then immediately place them inside an ice bath, and let them stand for 2 minutes.
Note: The samples should not be shaken or stirred during the let-stand period, since this might lower the transformation yield.
- Add 700μl of medium (LB Broth), without antibiotics, into the tube and mix. Afterwards, resuscitate the mixed cells by centrifuging them for 60 minutes at 200rpm and 37°C
-
Depending on the experimental needs, take the appropriate volume of the resuscitated cell suspension, spread it evenly onto the medium (LB Agar Plate) containing antibiotic Amp, and
place the agar plate upside down in a 37℃ incubator for overnight incubation
-
Prepare the PCR Reaction System
| Components |
Volume (50μl in total) |
| 2×T5 Super PCR Mix (Colony) |
25μl |
| 10μM M13-47 Primer |
2μl |
| 10μM M13-48 Primer) |
2μl |
| Template DNA (From one singular colony of competent cells; not yet added) |
2μl |
| ddH2O |
Added until the solution reaches 50μl |
- Use pipette tips to pick up a singular bacteria colony and insert it into the reaction solution. After blowing up and down to mix using the pipette, the tip is taken out
-
Perform the PCR Reaction as follows
| Temperature |
Time |
Cycles |
| 98℃ |
2 minutes |
1 |
| 98℃ |
10 seconds |
30 |
| 55℃ |
10 seconds |
| 72℃ |
10 s/kb |
| 72℃ |
2 minutes |
| 4℃ |
∞ |
|
- Detect positive colonies via gel electrophoresis
(1) Materials and Tools:
Easy Pure Plasmid MiniPrep Kit (EM101):
| Component |
EM 101-01 (50rxns) |
| Resuspension Buffer (RB) |
15 ml |
| Lysis Buffer (LB, Blue) |
15 ml |
| Neutralization Buffer (NB, Yellow) |
20 ml |
| Wash Buffer (WB) |
10 ml |
| Elution Buffer (EB) |
5 ml |
| RNase A (10mg/ml) |
150 μl |
| Mini-Plasmid Spin Columns with Collection Tubes |
50 |
NOTE: Before use, add the RNase A into the RB and store it at 2-8°C; Add 40 ml of 100% ethanol to WB, making the final ethanol concentration in WB to be 80%.
(2) Steps:
-
Take the positive cultures that have been incubated overnight, and centrifuge at 10000×g for 1 minute. Remove as much supernatant as possible. If the volume of the bacteria suspension
is too large, collect it by centrifugation several times
-
According to the following table, add the colorless solution RB (containing RNase A), and suspend the bacterial precipitate by shaking, so that no small pieces of bacteria should
remain
| LB Media |
RB |
LB |
NB |
| ≤5 ml |
250 μl |
250 μl |
350 μl |
| 5–10 ml |
500 μl |
500 μl |
700 μl |
| 10–15 ml |
750 μl |
750 μl |
1050 μl |
| 15–20 ml |
1000 μl |
1000 μl |
1400 μl |
-
According to the above table, add the blue solution LB. Gently invert the mixture up and down 4-6 times, so that the bacteria can be fully lysed. The color should change from
transparent to blue translucent, which indicates complete lysis (the process should not last more than 5 minutes)
-
According to the table above, add the yellow solution NB and mix gently 5-6 times (the color should change completely from blue to yellow, indicating uniform mixing and complete
neutralization) until firm yellow agglutinations are formed. Let stand at room temperature for 2 minutes
-
Centrifuge at 12000×g for 5 minutes. Carefully add the supernatant to a spin purification column. Centrifuge the column at 12000×g for 1 minute and discard the effluent. If the volume
of the supernatant is greater than 800pl, it can be added to the column in several portions and centrifuged as above one by one, discarding the effluent
- Add 650ul of Solution WB, and centrifuge at 12000×g for 1 minute, discarding the effluent
- Centrifuge at 12000×g for 1-2 minutes to completely remove the residual WB
-
Place the spin column in a clean centrifuge tube. Add 30-50pl EB or ddH2O (pH>7.0) to the center of the column and let stand at room temperature for 1 minute. (The EB or ddH2O should
be preheated in a 60-70°C water bath to ensure maximum effectiveness.)
- Centrifuge at 10000×g for 1 minute, elute DNA to collect the DNA solution and store at -20°C
(Note: This experiment requires aseptic operations on a laminar flow hood.)
- Add FBS serum, antibiotics (P/S, penicillin, streptomycin), and mycoplasma inhibitor to Gibco Schneider’s Drosophila Medium (1×) (referred to as S2 medium)
-
Retrieve the frozen S2 cells from the liquid nitrogen tank. Take precautions to prevent items from falling into the liquid nitrogen tank and to avoid frostbite from liquid nitrogen
- Quickly place the cryotube in a 37°C water bath for approximately 1 minute until completely thawed
- Transfer the culture fluid from the cryotube to a 1.5ml EP tube, centrifuge at 1500rpm for 10 minutes, and remove the supernatant
-
Add 500μl of S2 medium to the EP tube to resuspend the cells. Add 4.5ml of S2 medium to a 6cm dish, transfer the culture fluid from the tube to the culture dish, and gently swirl in a
zigzag pattern 8-10 times to mix the cells
- Seal the culture dish with parafilm, leaving a small gap. Place it in a CO2 incubator for cultivation
- After 3-5 passages following cell revival (depending on the cell's condition), proceed with subsequent experiments (transfection)
(Note: This experiment requires aseptic operations on a laminar flow hood.)
- After 3-5 days of S2 cell culture, when the cells cover the bottom of the culture dish and there is a certain density of suspended cells, passaging can be performed
-
Perform a 1:1 cell passage. Aspirate and mix 5ml of the primary cell culture fluid, take 2.5ml and transfer it to a new 6cm dish, and add 2.5ml of fresh S2 medium. Gently swirl in a
zigzag pattern 8-10 times to mix the cells
- Seal the culture dish with parafilm, leaving a small gap. Place it in a CO2 incubator for cultivation
(Note: This experiment requires aseptic operation in a biosafety cabinet.)
(1) Transfection Reagent:
Roche X-treme GENE™ HP DNA Transfection Reagent
(2) Steps:
Experiment 1: Transfection
-
3~5 days after the last subculturing (spreading a bacteria colony to two new cultures to ensure growth), perform cell seeding. Seed the bacteria to a 12-well plate, with the volume of
1ml per well (500μl primary cell culture medium + 500μl fresh S2 medium). The transferring method is the same as subculturing
-
24 hours after cell seeding, perform transfection. 1μg of plasmid should be transfected in each well of the 12-well plate, including pAc-Gal4 (250ng), pUAST-MTF-1 (375ng), and pMRE-Hid
(375ng). Each 1μg of plasmid should be diluted in 100μl of serum-free S2 medium. The ratio of plasmid to Roche transfection reagent was 1:2 (mass:volume, i.e., 1μg of plasmid
corresponded to 2μl of transfection reagent)
- Prepare the serum-free S2 medium, 1μg of 3 plasmids, and Roche transfection reagent as a mixture. Invert the container to mix, and let stand for 15 minutes
-
Add 100μl of the mixture to each well of the 12-well plate against the wall, into the culture solution. Shake 8~10 times in a cross shape (oscillate the plate vertically, horizontally,
then diagonally) to mix well
- Seal the 12-well plate mostly with sealing film, leaving a gap. Place the plate in a CO2 incubator
Experiment 2: Cell incubation and collection
-
48 hours after the cell co-transfection, add chemicals as experiment procedures. Add ZnCl2 and CdCl2 so that the concentration of Zn2+ and Cd2+ in four experimental groups of cells
were 10μM and 100μM respectively. ZnCl2 and CdCl2 had been already prepared as 1mM and 10mM storage solution, so add 10μl of the storage solutions into 1ml of the cell culture medium.
Cross shake 8~10 times to mix well and place it in a CO2 incubator for culture. (There should be 5 groups of cells in total: Control, 10μM Zn2+, 10μM Cd2+, 100μM Zn2+, 100μM Cd2+.)
-
4 hours after adding the heavy metal ions, collect the cells. Resuspend the cells through blowing them with a pipette, transfer each well of cell culture solution to a 1.5ml EP tube,
centrifuge at 1500rpm for 10 minutes, and aspirate the supernatant
- Temporarily freeze and store the cells at -80°C.
(Note: Avoid nuclease contamination during operation.)
- Add 500μl NucleoZOL to each tube of cells and blow to resuspend lysed cells
- Add 200μl DEPC water to the NucleoZOL and cell solution, mix well, and Vortex oscillate for about 15 seconds. Let stand at room temperature for 5 minutes
- Centrifuge at 12000×g for 15 minutes. Aspirate 500μl of the supernatant and transfer it to a new nuclease-free EP tube
- Add 500μl of isopropanol and mix through inversion. Let stand at room temperature for 10 minutes
- Centrifuge at 12000×g for 10 minutes. A white RNA precipitate should be visible at the bottom of the EP tube. Discard the supernatant and avoid aspirating the RNA precipitate
-
Wash with 500μl of 75% ethanol and centrifuge at 8000×g for 3 minutes. Discard the supernatant and avoid aspirating the RNA precipitate. Add 500μl of 75% ethanol and centrifuge at
8000×g for 3 minutes. Discard the supernatant and try to absorb the ethanol as much as possible without picking up the RNA precipitate
-
Open the lid and allow the precipitate to dry slightly. The drying time should not be too long or too short, and it should last until the RNA precipitate becomes translucent. Add 20μl
of DEPC water, Vortex shake for 3 minutes until the RNA is completely dissolved
- Determine the concentration and purity of the RNA solution. Store the RNA at -80℃
(Note: Avoid RNase contamination.)
(1) Reagents:
TransScript® Uni All-in-One SuperMix for qPCR
(2) Steps:
-
Perform on ice. Take an RNase-free 8-tube strip and prepare a 20μl reverse transcription reaction mix: Component Volume 20μl, Total RNA ≤1μg, 5×TransScript® Uni All-in-One SuperMix for
qPCR 4μl, gDNA Remover 1μl, and DEPC Water up to 20μl.
- Mix by pipetting and set up the PCR machine program: ZZY>TRANSRT. Incubate at 50°C for 5 minutes, deactivate at 85°C for 2 minutes, and hold
- Store the cDNA at -20°C
-
Add 200μl RIPA lysis buffer per tube (from one well of a 12-well plate). Prepare an appropriate amount of RIPA lysis buffer (strong) and add PMSF (100mM) to achieve a final
concentration of 1mM. (Perform on ice.)
- Add 200μl lysis buffer to each tube, resuspend the cells by pipetting, and let it sit on ice for 5 minutes
- Pre-cool the centrifuge to 4°C, and centrifuge the tube at 14000×g for 10 minutes
- Pipette 100μl supernatant into a new EP tube, add 25μl 5×SDS sample buffer, and mix by pipetting
Day 1:
- Sample Preparation (Protein Extraction).
- SDS-PAGE Gel electrophoresis (at 90V, room temperature for 2.5-3 hours)
- Transfer of proteins to membrane (at 25V, 4℃overnight)
Day 2:
- Ponceau Staining: to confirm the proper transfer of proteins from gel to membrane
- Block the membrane at room temperature for 1 hour
- Stain with primary antibody at room temperature for 10-12 hours
- Stain with secondary antibody at 4℃ for 2 hours
-
Develop and detect protein bands.
(Note: The specific experimental steps can be referred to Gogia N et al., 2017)
Reference:
Gogia N, Sarkar A, Singh A. An Undergraduate Cell Biology Lab: Western Blotting to Detect Proteins from Drosophila Eye, Lab Reports for Biology. Dros. Inf. Serv. 100 (2017): 218-225.
- Dissect 3rd instar larvae in solution A (50ml PBST + 50μl 1mM MgCl2 + 1.5ml 5M NaCl)
- Add acridine orange (AO) at a final concentration of 1×10-5 M for staining at room temperature for 5 minutes (avoid light)
-
Wash twice with solution A, quickly dissect and isolate the tissues required for the experiment, make slides, observe and take pictures under a fluorescence microscope
(Note: AO staining solution is toxic. Gloves should be worn during handling and light should be avoided.)
990μl buffer A +10μl AO (1×10-3 M), 5-minute fixation, dissect the discs out ASAP, and take photographs under fluorescence microscope.
- Dissect discs in cold PBS (800μl).
- Fixation: add 200μl 40% formaldehyde, and fix for 20 minutes
- Rinse: add cold PBS to rinse 3 times
- Dissect out discs, and make slides
- Microscopy
- Dissect discs in cold PBS (800μl)
- Fixation: add 200μl 40% formaldehyde, and fix for 20 minutes
- Rinse: add cold PBS to rinse 3 times
- Permeabilization: add 0.3% PBST for 20 minutes at room temperature, repeat 3 times
-
1st antibody : in a 0.6ml tube, 4℃ overnight
{
| 90μl |
0.3% PBST |
| 10μl |
5%BSA |
|
1st antibody |
- Rinse: 0.3% PBST to wash for 30 minutes, repeat 3 times
-
2nd antibody: room temperature for 2 hours
{
| 90μl |
0.3% PBST |
| 10μl |
5%BSA |
|
1st antibody |
- Rinse: 0.3% PBST wash for 30 minutes, repeat 4 times
- Mount: add a drop of mounting solution
- Microscopy