Team:Lethbridge/Wet-Lab/Protocols
Lab Protocols
Buffer Compositions
50 mM Tris-Cl
500 mM NaCl
0.2% Tween 20
5 % Glycerol
2 mM PMSF
5 mM β-mercaptoethanol
Add 10 mM to the imidazole and use for pre-equilibration and wash.
50 mM Tris-Cl
500 mM NaCl
0.2% Tween 20
5 % Glycerol
2 mM PMSF
5 mM β-mercaptoethanol
20 mM Imidazole
50 mM Tris-Cl
500 mM NaCl
0.2% Tween 20
5 % Glycerol
2 mM PMSF
5 mM β-mercaptoethanol
200 mM Imidazole
Preparation of Competent Cells
2. Inoculate from glycerol stock and grow cell culture overnight in 2 x 5 mL LB flasks.
- From Fabian: Do a streak plate the first night, then the overnight (2nd night) with 2x5ml per stock. 3rd day, start with the 5ml in 50ml and grow to OD 0.6
3. Centrifuge cells at 2 700xg for 7 minutes at 4°C.
4. Pour off supernatant being careful not to disturb the pellet. Tap gently on paper towel to remove any remaining supernatant.
5. Add 30 mL ice cold 80 mM MgCl2 20 mM CaCl2 to each tube. Resuspend by pipetting gently up and down. DO NOT VORTEX.
6. Centrifuge for 5 minutes at 2 700 x g.
7. Pour off supernatant.
8. Resuspend cell pellet in 2 mL ice cold 100 mM CaCl2.
9. Combine both tubes for a final volume of 4 mL.
10. Add 1.2 mL sterile glycerol and mix gently.
11. Aliquot samples (20 – 100 µL each), flash freeze with liquid nitrogen and store at -80°C.
Transformation
2. Add 1 μL of DNA to 25 μL of competent cells.
3. Incubate on ice for 30 minutes.
4. Incubate at 42°C for 45-60 seconds
5. Incubate on ice for 5 minutes.
6. Add 400 μL of LB media.
7. Incubate while shaking at 37°C for 1 hour.
8. Pour culture on agar plates, spread plate and leave right side up for 30 minutes in the incubator.
9. After 30 minutes, put the plate upside down in an incubator at 37°C overnight.
Dual Transformation
- Thaw for approximately 30 min.
2. Add the calculated amount of each sample of DNA to 50 μL of competent cells.
- 0.28 μL of P22C2-1 and 0.35 μL SPCas9C1, in this case.
3. Incubate in ice-water slurry for 30 minutes.
- Set water bath to 42°C during this time.
4. Incubate at 42°C for 45-60 seconds.
5. Incubate in ice-water slurry for 5 minutes.
6. Add 950 μL of LB media.
- SOC medium is ideal if available
7. Incubate in a shaking incubator at 37°C and 250 rpm for 1.5 hours.
8. Pour ALL culture on agar plates, spread plate and leave right side up for 30 minutes in the incubator.
9. After 30 minutes, put the plate upside down in an incubator at 37°C overnight.
Overexpression and SDS-PAGE
2. Pellet cells at 5000 g for 7 minutes using a 50 mL plastic falcon tube (multiple spins).
3. Remove supernatant and resuspend the pellet in 10 mL fresh LB.
4. Check the OD600 of the mixture (will need a 1000-fold dilution for the UV spectrophotometer linear range 0.1-1).
5. Inoculate the expression flasks (2L flasks 500 mL LB) to an OD600 of 0.1 with the overnight mixture.
6. Add appropriate antibiotic to the expression cultures and incubate at 37ºC with shaking.
7. Take OD600 readings from 1 flask every ½ hour to establish a growth curve.
8. When the expression cultures reach a 0.6 OD600 take a 1 OD600 sample (T0) and induce the culture with IPTG to a final [IPTG] of 1 mM IPTG.
9. Take 1 OD600 samples every hour for 3 hours (T1-T3) and OD600 readings every ½ hour. Dilute samples appropriately when the OD600 becomes more than 1.
10. Harvest cells in the JA-14 rotor at 5000 g (5700 rpm) for 10 minutes using multiple spins.
11. Discard supernatant in the bacterial waste and scrape the cells into a clean sterile 50 mL falcon tube and rinse out the centrifuge tubes using LB to wash off remaining cells. Pour LB into the 50 mL falcon tube and centrifuge at 5000 g for 10 minutes.
12. Discard supernatant weigh cell pellet and freeze with liquid nitrogen. Store pellet in the -80ºC freezer until ready to purify protein.
13. Open the samples with 100 µL of cell opening buffer and heating to 95°C. Add 10 µL of SDS and pellet the debris by microcentrifugation at max speed for 1.5 minutes.
14. Prior to loading on the SDS gel heat the samples to 95°C for 5 minutes.
15. Load 10 µL of the sample on a 10-15% acrylamide gel and run at 200V for ~1 hour.
16. Stain the gel for 30 minutes with shaking using, SDS staining solution and then destain overnight with SDS destaining solution. Faster/better destaining can be achieved by including a folded wad of paper towel to the destaining vessel to soak up the dye.