Equipment
Reagents
SpII:
- 200ml T base,
- 50% (w/v) glucose 2ml,
- 1.2% (w/v) MgSO4.7H2O,
- 1% (w/v) casamino acids 2ml,
- 10% (w/v) bacto yeast extract 2ml,
- 0.1M CaCl2 1ml,
- Antibiotic growth selectors.
T base per litre:
- 2g (NH4)2SO4,
- 18.3g K2HPO4.3H2O,
- 6g KH2PO4,
- 6g trisodium citrate 2H2O
SpC:
- 20ml T base,
- 50%(w/v) glucose 0.2ml,
- 1.2% (w/v) MgSO4.7H2O 0.3ml,
- 10% (w/v) Bacto yeast extract 0.4ml,
- 1% (w/v) casamino acids 0.5ml,
- growth selectors
SpII + EGTA:
- 200ml SpII,
- 4ml EGTA (0.1M, pH 8.0,0.152g),
- Store in frozen small aliquots (20ml),
Protocol
Day 1
- Inoculate 20ml of LB media in a 200ml flask with a single colony from an LB agar plate of B. subtilis and grow overnight at 30oC.
Day 2
- Remove 0.5ml of the overnight solution and place in a cuvette, add 1ml of LB media and mix thoroughly before measuring the O.D.600 of the solution using LB media as a blank. This dilution is essential because the relationship between O.D.600 and cell number is only linear up to an O.D.600 of 1.5, overnight cultures typically reach an O.D.600 of 2-3.
- Using the following calculation work out the dilution required to get an OD600 of 0.5 in 20ml:
- Volume of Overnight Culture (X) = (0.5/O.D.600)*20ml
- Add the required volume of overnight solution to 20ml of pre-warmed SpC medium in a 200ml flask. Incubate this culture at 37ºC with vigorous aeration (200rpm) and take periodic O.D.600 to assess cell growth. It is important to remember if the O.D.600 is greater than 1.5 we must dilute the cultures down and then multiply the O.D.600 back up.
- When the rate of cell growth is seen to depart from exponential (i.e. no significant change in cell density over 20-30 minutes, occurs at 2.0-2.5) inoculate 200ml of pre-warmed SpII medium with 2ml of stationary-phase culture and continue incubation at 37ºC with slower aeration (150rpm).
- After 90 minutes incubation, pellet the cells by centrifugation (4000rpm, 15 minutes) at room temperature. Typically to do this separate the 200ml cultures into 6 x 50ml falcon tubes.
- After centrifugation carefully remove the supernatant and save at least 20ml of it in a sterile 50ml falcon tube. The pellets are very small and so it is essential to remove the supernatant immediately after spinning and also very carefully. Resuspend the pellets in 18ml of the saved supernatant and add 2ml of sterile glycerol; mix gently.
- Aliquot the competent cells (0.5ml) in sterile eppendorf tubes and freeze rapidly in liquid nitrogen or a dry-ice and store at -80ºC.
Day 3
- Thaw competent cells rapidly by immersing frozen tubes in a 37ºC water bath,
- Aliquot cells into 200μl samples in sterile eppendorf tubes (200μl for each condition used). Add 200μl of SpII and EGTA and mix.
- Separate out each 400μl mixture back into 200μl samples (given 2 repeats for each condition).
- To each of these 200μl samples add suitable levels of DNA - between 10ng-500ng seems reasonable, and incubate in a shaking incubator at 37oC for 30 minutes (10 minutes required for integration)
- Remove the samples from the incubator and add 20μl of x10 Tbase and 2μl of 5% glucose solution. Mix thoroughly and then aliquot the mixture onto an LB agar plate containing suitable antibiotics. Under sterile conditions, spread out this mixture until dry. Tip - Do not turn the plates up side down for 10 minutes or so to let the solution completely dry off.
- Place in a 30oC incubator
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