Adapted from the procedure in Zhou 2009
Transformation of pET29b-BglB into E. coli K12 CJ236 FΔ(HindIII)::cat (Tra+ Pil+ CamR)/ ung-1 relA1 dut-1 thi-1 spoT1 mcrA
- chem competent CJ236 cells
- WT BglB plasmid
- selection plate containing 1X kanamycin and 1X chloramphenicol
- 15 mL round-bottom Falcon tube
- waste bucket
- P200, P1000 and tips
- Transform as in
production.md - Make media for Day 2: autoclave 2, 250 mL flask with 50 mL TB
- Plate and incubate overnight at 37 C
ssDNA Production (growth, virus inoculation, and overnight)
- M13 phage (NEB)
- media made yesterday, 2, 250 mL flask with 50 mL TB
- 2, 15 mL round-bottom tubes
- waste bucket
- P20 and tips
- serological pipet aspirator
- 50 mL serological pipet
- Dispense 3 mL into each tube
- Scrape 6 colonies into each tube
- Grow for 4-6 hours at 37 C with shaking until cloudy
- Add 3 µL of M13K07 phage
- Continue growing for 1 hour at 37 C with shaking
- Expand culture by diluting 1 mL from the tubes into the media in the flasks
- Grow overnight at 37 C with shaking
Harvesting ssDNA and evaluation of the procedure
The phage particles produced in a 50 ml overnight culture of JM109/pGEM5Z(+)-EGFP/M13KO7 complex were recovered from the supernatant by the classical PEG precipitation procedure [6] and lysed in 2 ml of a disruption buffer (5 M Guanidinium Hydrochloride, 1% Triton X-100, and 10 mM MOPS, pH 6.5) with a short incubation of 5 min at room temperature. A silica column (Tiangen Biotech Co., LTD, Beijing, China) with a maximum DNA adsorption quantity of 500 μg was placed inside a 50-ml Falcon tube. The cell lysate was poured into the column, which was then centrifuge at 10,000g for 2 min. The flow-through solution was discarded and the column was washed twice with 5 ml of 80% isopropanol and centrifugation at 10,000g for 2 min. The column was then transferred to a new Falcon tube and incubated with 1 ml of sterile TE buffer for 2–3 min to elute the bound DNA. The eluted DNA in the TE buffer was collected by centrifugation at 10,000g for 2 min. The 50 ml culture usually yielded a total 150–200 μg ssDNA
| Amount | Material |
|---|---|
| 2 | 50 mL Falcon tube |
| 2 | 2 mL microcentrifuge tube |
- software
- waste bucket
- serological pipet aspirator
- P20, P200, P1000 and tips
- hardware
All centrifugation steps at 4 C
- Spin down overnight culture in 50 mL tube at 4700 RPM for 20 min
- Aliquot 10 mL salt PEG to fresh 50 mL tubes
- Transfer supernatant (contains phage) to fresh tubes
- Vortex and incubate on ice for 45 min
- Spin down the phage at 4700 RPM for 45 min
- Decant liquid and let tube stand upright to drain off the rest of the liquid
- Resuspend the pellet in 2 mL PBS by vortexting or pipeting
- Spin at 14,000 RPM for 5 min
- Transfer the supernatant to new microfuge tubes with 300 µL salt PEG
- Vortex and incubate at room temperature for 10 min
- Spin down phage at 14,000 RPM for 2 min
- Pipet supernatant off pellet
- Do a second quick spin to collect residual liquid and pipet it off
- Resuspend the pellet (phage) in 1 mL PBS
- Spin down at 14,000 RPM for 5 min
- Set centrifuge to 8,000 RPM
- Transfer the supernatant (phage) to a new microfuge tube
- Purify using Qiagen M13 kit, do not spin over 8,000 RPM
If A260 implies DNA concentration is below 20 ng/µL the procedure likely didn't work. Run a gel to be sure, or just try again.
Run a gel to make sure you can see a clear band. The pET29-BglB construct is ~6700 bp. Single-stranded DNA runs about 10% faster on a gel than supercoiled double-stranded DNA.
- 200g PEG (polyethylene glycol) 8000MW
- 141.6g NaCl
- Add dH20 to 1L
- Autoclave with a stir bar, stir immediately after autoclaving until cool, and store at 40C.
- 800 mL water
- 8g NaCl
- 0.2g KCl
- 1.44g Na2HPO4 sodium phosphate
- 0.24g KH2PO4 potassium phosphate
- Adjust pH to 7.4
- Add water to 1 L
- 5 M Guanidinium Hydrochloride
- 1% Triton X-100
- 10 mM MOPS
- pH 6.5