Day 1: Starter Culture. Start procedure in the afternoon.
1. Warm growth plates. Thaw amp.
2. Add ampicillin 100ug/mL to LB. Array LB broth 1 mL/well into your deepwell plate.
3. Using a multichannel pipet tip, select a single colony from each agar plate square, and inoculate the appropriate well the growth plate. Leave tip in. Repeat on a new plate, so you have an A and B clone.
4. Leave tips in, and cover with breathable sealing tape. Incubate overnight 37°C, shaking.
Day 2: Induction
1. Noon: make M9 media.
979 mL water
10 mL mineral supplement solution
10 mL 50% glycerol solution
1 mL Kan + Amp + vitamin solution
1 pouch of M9 salts
1 L total
2. Array 1mL/well into a deepwell plate. This is your induction plate.
3. Inoculate the induction plate with 40µL of your starter culture. Centrifuge remaining starter culture at 1700rcf for 15 minutes. Dump supernatant, freeze pellets for later plasmid purification.
4. Incubate at 37°C, shaking, for 4 hours until the OD600 is 0.8 -1.
5. Turn temperature down to 4°C, incubate for 30 min.
6. Mix 40µL of induction plate with 40µL of 50% glycerol. Freeze this glycerol stock.
7. Add 30µL of IAAC + Sel-Met + IPTG solution. Grow overnight at 18C.
Day 3: Harvest & Lysis
1. Centrifuge your induction plate at 1700rcf for 15 minutes; discard supernatant and freeze pellets.
2. Prepare lysis buffer:
15 mL of 4X lysis buffer
45 mL of water
300 µL of 1M DTT
1 tablet of Roche protease inhibitor
Mix thoroughly, then add:
20 µL of rLysozyme and Benzonase
3. Add 125 µL of lysis buffer to each well of your frozen cell pellets.
4. Vortex thoroughly, until all pellets are resuspended.
5. Incubate at 25C, shaking, for 30 min.
6. Freeze on dry ice, 15 min.
7. Sonicate frozen plates for 1.5 min each, at around 250W. Keep water bath as cold as possible.
8. Freeze-sonicate plates (as in steps 4 and 5) twice more.
9. Incubate at 25C, shaking, for 30 min.
10. Freeze-sonicate plates (as in steps 4 and 5) three times.
11. Remove 10uL of sonication mixture, and mix with 30 µL of SDS loading buffer. Boil and freeze. This is your expression sample.
12. Prepare TEV digestion mix:
0.5 mL 4X lysis buffer
1.5 mL water
10 µL 1M DTT
200 µL 0.5M EDTA
3 mL TEV crude extract
13. Add 10uL of TEV digestion mix to 90 µL of sonication mixture in a new conical plate.
14. Spin down at 1700rcf, (do not dump supernatant though) leave at 4C overnight.
1. Spin down at 1700rcf. Remove 40 µL of the supernatant (make sure not to get any of the pellet). Add to 20 µL of 4X SDS loading buffer. Boil and freeze. This is your solubility sample.
Overview: Purify plasmids from expressing clones, for storage and future transformation.
Hazards: Normal precautions. Procedure includes alkaline, acidic, combustible, and irritant solutions.
Materials: - Frozen pellets from starter cultures.
- New deepwell plates.
- Buffers: P1, P2, N3, PE, TE (or EB).
- Turbo Filter and QiaPrep Filter.
- New skirted plates.
Notes: Use cone shape deepwell plates only.
1. Resuspend frozen pellets in 150 µL of Buffer P1. Vortex thoroughly.
2. Use Biomex FX to combine the best-expressing clones into one plate.
3. Add 150 µL of Buffer P2. Shake gently, do not vortex. Add N3 after 5 minutes. This timing is important here… try to get it right around 5 minutes.
4. Add 250 µL of Buffer N3. Shake gently, do not vortex.
5. Incubate on ice for ~20 minutes, to allow greater precipitation of ssDNA, KDS, etc.