Bacillus subtilis transformation methods

R. Jayakumar jakku at mrna.tn.nic.in
Wed Nov 15 11:09:37 EST 2000


what about the natural competency method which I have been using. It is
fairly common and can be found in any book. Anyway here it is

 B. subtilis becomes naturally competent thus obviating the need to use
standard E. coli transformation procedures.

 Grow 3 ml culture in spC at 37 degrees for 5 hours
Add culture to 27 mls of SpII and continue incubation at 37 degrees
 for 90
 minutes
 Pellet cells (IMPORTANT:  save 1600 ul of supernatant as this contains
 the "competence factors")
 Resuspend the cells in the 1600 ul supernatant
 Add 400 ul 50% glycerol and aliquot as 500 ul
 Freeze at -80 until use

 For transformation: Thaw tube of comp Bacillus
Add equal vol SpII + 2 mM EGTA
 Add DNA (1-10 ul) and incubate at 37 degrees for 20-30 minutes
 Plate cells on selective media


10X T-Base:  150 mM ammonium sulfate, 800 mM K-phosphate (dibasic), 440
K-phosphate (monobasic), 35 mM Na-Citrate

SpC:  100 mls 1X T-Base
      1.0 ml  50% Glucose
      1.5 mls 1.2% MgSO4
      2.0 mls 10% yeast extract
      2.5 mls 1% case amino acids

SPII: 100 mls 1X T-Base
      1.0 ml  50% Glucose
      7.0 mls 1.2% MgSO4
      1.0 ml  10% yeast extract
      1.0 ml  1% case amino acids
      0.5 ml  100 mM CaCl2

SPII-EGTA:  SPII + 2 mM EGTA


NOTE>  Can you send me the protocol of Spizizen. for electroporation.  I
have also been trying out the Matsuno's electroporation protocol for B.
subtilis, but to no avail.

bye
jakku
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R. JAYAKUMAR
CSIR- Senior Research Fellow
Dept. of Molecular Microbiology,
School of Biotechnology,
Madurai Kamaraj University,
Madurai - 625 021.
India
email: jakku at linuxfan.com
tel: +91-452-858471-374
efax: (603)-688-4665
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"YOu harvest what you sow.. so sow happiness everywhere"
----- Original Message -----
From: Andreas Pfoestl <andreas_pf at hotmail.com>
To: <methods at hgmp.mrc.ac.uk>
Sent: Tuesday, November 14, 2000 3:40 PM
Subject: Bacillus subtilis transformation methods




> Hello!
>
> We are trying to transform different Bacillus subtilis strains and are
> seeking alternative protocols to the method of Spizizen (e.g.
> electroporation).
> Is anyone familiar with such methods to help me out?
>
> Thank you very much
> Andreas
>
>
>


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