Cfrench:BacTrans1: Difference between revisions
From OpenWetWare
Jump to navigationJump to search
(New page: ==Transformation of ''Bacillus subtilis''== This protocol was developed by Lucy Montgomery based on published protocols (Anagnostopoulos and SPizizen, 1961, J. Bacteriol. '''81:'''741 and ...) |
(No difference)
|
Revision as of 09:46, 19 October 2007
Transformation of Bacillus subtilis
This protocol was developed by Lucy Montgomery based on published protocols (Anagnostopoulos and SPizizen, 1961, J. Bacteriol. 81:741 and Young and Spizizen, 1961, J. Bacteriol. 81:823). It has been used successfully with pVK168 (specR) and pTG262 (cmlR).
Materials needed
- Plate of B. subtilis 168
- 20% glucose
- 1.0 M MgSO4.7H2O
- 0.1 M CaCl2
- 0.05 M MnSO4
- 20% w/v casamino acids
- 11 mg/ml indole (dissolve in dimethyl sulfoxide)
5 x Spizizen's Salts
- available from the media prep room; if you need to make this for yourself, it contains:
- 1% w/v ammonium sulphate
- 7% w/v K2HPO4
- 3% w/v KH2PO4
- 0.5% w/v Na3citrate dihydrate
- 0.1% w/v magnesium sulphate)
Solution P
- 25 ml 1 M MgSO4
- 5 ml 0.1 M CaCl2
- 0.1 ml 0.05 M MnSO4
- 70 ml water
Pre-Transformation Medium (PTM)
- 2 ml 5 x Spizizen's Salts
- 8 ml water (sterile)
- 0.5 ml 20% glucose
- 0.1 ml solution P
- 0.2 ml 20% casamino acids
- 0.01 ml indole (11 mg/ml)(B. subtilis 168 is auxotrophic for tryptophan and requires indole or tryptophan to be provided).
Transformation Medium (TM)
- 2 ml 5 x Spizizen's Salts
- 8 ml water (sterile)
- 0.3 ml 20% glucose
- 5 microlitres 20% Casamino Acids
- 10 microlitres indole (11 mg/ml)
Procedure
- Grow an overnight culture of B. subtilis 168 in L-broth.