Ø Culture Mediums preparation
Ø PCR
Ø Plasmid Extraction
Ø& E. coli Transformation
ØAgarose gel Electrophoresis
Ø Enzyme digestion
Ø Enzyme ligation
Ø Protein Purification and Concentration
Ø SDS-PAGE
Ø Function Test: BRET
* Samples were sent to SubCat for IP-MS test and analysis thus no IP-MS procedures will be listed below.
2）Apparatus
Preparatory Work
Configuring LB Solid Medium
|
|
Name |
|
1 |
250mL Erlenmeyer Flask |
|
2 |
Drug Spoon |
|
3 |
Weighing Paper |
|
4 |
Electronic Weight |
|
5 |
Petri Dish |
|
6 |
Sealing Film |
|
7 |
500mL Measuring Cylinder |
Culturing Escherichia Coli
|
1 |
Single Channel Pipette |
|
2 |
Pipette Tips |
|
3 |
Centrifugal Tube |
|
4 |
Incubator Shaker Series |
Centrifuging E.coli in LB Broth
|
1 |
Micro Centrifuge |
|
2 |
Micro centrifuge tubes |
|
3 |
Incubator shaker series |
|
4 |
Purification column |
|
5 |
Outer thimble |
|
6 |
Pipette tips |
|
7 |
Single Channel pipette |
Plasmid Construction
Plasmid Enzyme digestion
|
1 |
Micro centrifuge tube |
|
2 |
Single channel Pipette |
|
3 |
Pipette tips |
|
4 |
Ice tube |
|
5 |
Thermostat Water Bath Cauldron |
Agarose gel electrophoresis and recycling
|
1 |
Centrifuge tube |
|
2 |
Erlenmeyer flask |
|
3 |
20/200μL Single channel Pipette |
|
4 |
Pipette tips |
|
5 |
Microwave Oven |
|
6 |
Electronic Balance |
|
7 |
Electrophoresis tank |
|
8 |
Comb |
|
9 |
High channel level electrophoresis apparatus |
|
10 |
Imagine System |
|
11 |
Eppendorf Micro Test Tubes |
|
12 |
UV Imaging System |
|
13 |
Scalpel |
action
|
1 |
Micro Centrifuge Tube |
|
2 |
DNA Purification Column |
|
3 |
14mL Polypropylene Round-Bottom Tube |
|
4 |
Centrifuge |
|
5 |
200µL/1mL Single Channel Pipette |
|
6 |
Pipette Tips |
|
7 |
Vortex Oscillator |
|
8 |
NANODROP 2000 |
Enzymatic digestion
|
1 |
Centrifuge |
|
2 |
Pipette tips |
|
3 |
1mL/200μL/2μL Single Channel Pipette |
|
4 |
NANODROP 2000 |
|
5 |
Thermostat Water Bath Cauldron |
|
6 |
Electrophoresis System |
|
7 |
Ice Tube |
|
8 |
Eppendorf Micro Test Tubes |
|
9 |
Scalpel |
|
10 |
UV Transilluminator |
Colony PCR
|
1 |
Thermal Cycler |
|
2 |
Benchtop |
|
3 |
Single channel pipette |
|
4 |
Pipette tips |
|
5 |
Micro centrifuge tube |
|
6 |
Ice Tube |
|
7 |
Benchtop High Speed Refrigerated Centrifuge |
|
8 |
Culture Dish |
BRET
|
1 |
Single Channel Pipette |
|
2 |
Pipette Tips |
|
3 |
Micro Centrifuge Tubes |
|
4 |
6 Well Cell Culture Plates |
|
5 |
Microplate Reader |
|
6 |
ELISA Microwell Strip Plate, 96-Well, black |
Product acquisition
Cell transfection
|
1 |
Fluorescent Inverted Microscope |
|
2 |
Electrothermal Thermostatic Bath |
|
3 |
Air-jacketed CO2 Incubator Panasonic |
|
4 |
Cell Counters |
|
5 |
Benchtop |
Protein purification
|
1 |
Single Channel Pipette |
|
2 |
Pipette Tips |
|
3 |
Centrifuge Tubes |
|
4 |
Small Vertical Electrophoresis Tank |
|
5 |
Comb |
|
6 |
Glass Pane |
|
7 |
Glue Mold |
3）Procedure
Preparatory Work
Preparation of LB agar Plate
1. Weigh 2.5g LB broth and 1.5g agar powder add to a 250mL Flask.
2. Measure out approximately 100 mL of deionized double distilled water(ddH2O) and add to Flask.
3. Use a sealing film to seal the Erlenmeyer flask
4. Mix until powder is dissolved.
5. Label the date, sample name, and group number on the flask and then Autoclave sterilization at 121℃.
6. Add antibiotic to the culture medium and mix well in the aseptic laboratory benchtop
7. Pour the culture medium into the Petri dish in the aseptic laboratory benchtop.
Culturing Escherichia Coli (E. coli)
1. Prepare LB liquid culture medium.
2. add Venus-K-Ras plasmid (Kan+), GST-α2A-AR-CT plasmid(Amp+), α2A-AR-Rluc8 plasmid(Amp+）to different tubes with LB liquid medium
3. Mark the name of different tubes
4. Put these tubes into in incubator shaker series overnight
Plasmid Construction
PCR preparation
(1) PCR system
|
PCR systems |
|
|
Reagent |
Volume（μL） |
|
2× PCR Mix |
25 |
|
Primer F (10 μM) |
2.5 |
|
Primer R (10 μM) |
2.5 |
|
DNA template |
2 |
|
ddH2O |
18 |
|
Total |
50 |
(2) PCR Program :
1. 95 ℃ 3 min;
2. 95 ℃ 30 s
3. 56 ℃ 30 s
4. 72 ℃ 2 min; 2-4 for 30 cycles
5. 72 ℃ 5 min
Plasmid Enzyme digestion
1. Transfer the plasmid solution into new centrifuge tubes, 30 μL each.
2. Add 10 μl water into each new centrifuge tube.
3. Add 5 μl green buffer into each new centrifuge tube.
4. Add 2.5 μl fast digest Xba1 enzyme into each new centrifuge tube.
5. Add 2.5 μl fast digest Sba1enzyme into each centrifuge tube.
Mix these samples and put the tube with the mixture into the 37°C water bath for 1.5h.
Purification target DNA
1. Weigh 1% Agarose, 0.5g on a scale.
2. Add the Agarose into a flask.
3. Weigh 50mL of TAE buffer using a graduated cylinder
4. Add weighted TAE buffer into the flask.
5. Fully mix the TAE Agarose solution.
6. Place the flask into a microwave and heat the solution until Agarose fully dissolves.
7. Place separating comb on the gel tray.
8. Pour the mixture into a gel tray.
9. Add 5 μL of YeaRed nucleic acid gel stain using a pipette.
10. Wait for the gel mixture to solidify.
11. Carefully remove the gel from gel tray and place it into an electrophoresis apparatus.
12. Add TAE buffer until the solution fully covers the gel.
13. Load previous PCR sample using a pipette into the sample holes created by the separating comb.
14. Electrify solution with a negative charge on the left and a positive charge on the right to make the solution flow from left to right. 140V 40min
Agarose gel electrophoresis and Gel Extraction
1. Put the Dual Sided Comb on the gel board
2. Weigh and add 0.25g of agarose into the flask
3. Add 25mL of 1×TAE into the flask
4. Heat the mixture with a microwave oven to let the agarose completely dissolved in the TAE
5. Add 2.5μL of YeaRed into the flask
6. Pour the dyed solution onto the gel tray and cool it
7. Take the dual sided comb out of the gel tray
8. Take the casting tray out of the gel tray
9. Put the tray on the side that is near to the cathode of the gel box
10. Add all of the 3 samples into each corresponding groove of the gel
11. Add 5-10μL of marker into one of the grooves of the gel
12. Close the gel box and start the electrophoresis with 160V for 30 minutes
13. Take the gel out and put it under the imaging system
14. Identify each DNA strand after the gel electrophoresis
15. Excise the DNA fragment from the agarose gel with a clean, sharp scalpel
16. Put each gel slice into the centrifuge tube and add 500μL Buffer B2 to each tube
17. Incubate at 50°C for 10 min
18. Transfer the binding mix into the miniprep column
19. Centrifuge the miniprep column at 8000rpm for 30 seconds and then discard flow-through
20. Add 500μL of wash buffer to the miniprep column and centrifuge at 9000rpm for 30 seconds and discard flow-through
21. Repeat step 20
22. Place the miniprep column back into the same tube and centrifuge for 1 minute at 9000rpm, then place miniprep column into a clean 1.5 ml microcentrifuge tube
23. Add 15-40μL of EB to the center of column membrane and let the column stand for 1 min in room temperature, and then centrifuge for 1 min.
Gel DNA Purification Kit and enzyme digestion
1. Weigh the target gel
2. Add 3-6× buffer B2
3. Water bath the solution at 50℃ until the gel is completely dissolved
4. Combine miniprep column and collection tube and transfer the binding mix to the column
5. Place the tubes into a centrifuge (12000rpm 28℃, 30sec)
6. Pour out the discard solution and add 500μL wash solution
7. Place the tubes into a centrifuge (12000rpm 28℃, 30sec)
8. Repeat steps 6-7
9. Pour out the discard solution and put it in the centrifuge (1200rpm, 28℃, 2mins) without adding anything
10. Change a collection tube and add in 30μL ddH2O
11. put it in the centrifuge (1200rpm, 28 degrees, 2mins)
12. Collect the solution in the collection tube and add it back to the column
13. Repeat step 11
14. Label the solution in the tube
15. Measure the concentration of the DNA solution
16. Use an ice tub to contain the enzymes
Add enzyme, buffer solution*2, H2O into the PCR solution
T4 ligase link target gene and vector and junction product transformation
1. Add 1 µL T4 DNA Ligase Buffer, 3µL XbaI-SdaI α2A-AR-Rluc8, 5µL XbaI-SdaI α2A-AR-Rluc8-vector, 1µL T4 DNA Lisase to the PCR tube and mix well.
2. Put the sample into the PCR instruments and set up in 25℃ for 1 hour
3. Add 10µL mixture of T4 carrier and purpose gene to DH5α competent cell and put on ice for 25min
4. Add 1µL GST- α2A-AR-CT plasmid to BL21(DE3) chemical competent cell and put on ice for 25min
5. Heat shock 90 seconds at 42 °C in H2O bath.
6. Put these samples on ice for 2min
7. Add 700µL LB without resistance into the sample
8. Put these sample into water-soluble pot as 37°C for 30min
Transformation of E. coli by heat shock and spread plate cultivation
1. Take 50μl each of BL21(DE3) and DH5α and place each of them into two test tubes.
2. Add 1μl of GST- α2A-AR-CT Plasmid to BL21(DE3) competent cells and 10μl of ligation product to DH5α.
Colony PCR
1. Divide the solution into 10 equal parts and replace them in 10 separate tubes.
2. Using a pippete tip, move parts of the bacteria colony into centrifugal tubes （in a benchtop）
3. Put the tubes into the thermal cycler for 95℃ for 3 minutes. 95℃ for 30 seconds,55℃ for 30 seconds, and 72℃ for 2 minutes. This prodecdure should be repeated for 32 times.
4. Place the tubes at 72℃ for 5 minutes and store them at 12℃.
Plasmid Extraction
1. Add 500µl of Buffer S into each of the 4 collection tubes with Miniprep columns
2. Centrifuge these tubes for 30 seconds at 12000×g
3. Add 250µl of buffer SP1 into the tubes
4. Make the bacteria in the tubes floating using a shaker
5. Move the bacteria into the collection tubes.
6. Add 250µl of buffer SP2 into the collection tubes
7. Add 350µl of buffer SP3 into the collection tubes and invert immediately but gently 4–6 times.
8. Centrifuge these tubes for 10minutes at 8000×g
9. Transfer the cleared lysates to the new collection tubes
10. Centrifuge these tubes for 30 seconds at 8000×g
11. Discard flow-through
12. Add 500µl Buffer DW1 into each of the 4 collection tubes
13. Centrifuge these tubes for 30 seconds at 9000×g
14. Add 500µl wash solutions into each of the 4 collection tubes
15. Centrifuge these tubes for 30 seconds at 9000×g
16. Discard flow-through
17. Add 500µl wash solutions into each of the 4 collection tubes
18. Centrifuge these tubes for 60seconds at 9000×g
19. Discard flow-through
20. Move the column into a centrifuge tube
21. Add 35-50µl of Elution buffer into the tube, centrifuge for 1 min.
Protein Purification
Induction of bacteria and Protein induced expression
1. Transform the α2A-AR-CT plasmid into E. coli BL21(DE3)
2. Inoculate the strains into 5mL LB medium, incubate overnight
3. Transfer 0.5 mL of bacterial solution into fresh LB medium (100 mL)
4. incubate until OD600=0.5
5. Add IPTG (1 mM) and incubate 37 ℃, 220 rpm overnight
Protein purification
1. Centrifuge and collect the bacteria at 4,000 rpm for 5 minutes, discard the supernatant.
2. Mix the strain with 4ml lysis buffer and then Ultrasonically lysed bacteria on ice (500W, 3s/3s, 10 min).
3. Centrifuge at 10,000 rpm at 4℃ for 20 min, and collect the supernatant.
4. Add 1 mL mixed well GST-tag purification resin, centrifuge at 1000g for 1min, discard the supernatant. Mix the resin with 0.5 mL lysis buffer and transfer the mixture to the column.
5. Add the supernatant to the column and flow through, repeat for 5 times.
6. Add 1 mL lysis buffer to the column and collect the fluid. Repeat for 4-5 times.
7. Collect the sample for SDS-PAGE.
SDS-PAGE
1. Mix the protein solution with 6× loading buffer
2. Place it in the 100℃-water bath for 5 minutes
3. Extract 20 μL of the product for SDS-PAGE electrophoresis
4. Stain the gel using Coomassie Blue Staining Solution, then destain it using Destaining Solution overnight.
5. Blots were imaged using an LI-COR Biosciences Odyssey imager.
Product acquisition
HEK293 Cell culture and transfection
1.Culture the HEK293 cell in DMEM culture medium at 37℃ 5% CO2 overnight.
2. Change the DMEM culture medium used to cultivate HEK293 cell into each of the six platforms culture plate
3. add 150µL of DMEM, Str KDEL_ SBP-α2A-AR-Rluc8 plasmid 250ng, Venus-K-Ras plasmid 750ng , and 3µL of PEI into each of the 6 centrifuge tubes
4. pipette up and down to mix solution
5. Put the 6 centrifuge tubes into an incubator for10 minutes
6. Transfer the solution in each centrifuge tube to its corresponding platform culture plate
Cell passage
1. Aspirate cell media from culture dish.
2. Wash dish with 5mL PBS
3. Aspirate PBS
4. Add 2mL of Trypsin-EDTA to digest the cells
5. Add 6mL of DMEM in order to neutralize typsin-EDTA activity
6. Count cell number, then compute the volume of the DMEM we need to add.
7. Add 13.3mL DMEM into the petri dish to dilute the cell liquid
8. Add 3mL of the diluted liquid into each cell in the six-well cell culture plate
9. Place the six-well cell culture plates in the incubator at least 16 hours
α2A-AR RUSH-BRET
1. Add 40µL of Biotin to each well and incubate for 0, 15, 30, 1, and 3 hours. The group without biotin for 0 minutes was used as the control group.
2. Discard the culture medium, wash the cells twice with DPBS, add 1ml of DPBS to resuspend cells.
3. Remove a 200 μL sample of suspension to three wells in a black opaque 96-well plate and wait for 15min.
4. Add 2µL Coelenterazine H to these 3 wells.
5. Put the plate into the ELISA Microplate Reader and record the value.
