"false transformants" from B. subtilis transformation - how to get rid of them? (May/07/2009 )
Hi there!
I've been trying to transform Bacillus subtilis with a plasmid carrying kanamycin resistance to knock out a gene. I use Spizzizen's protocol. My problem is, my "cells only" control (no plasmid added) keeps growing on the TBAB+kan plates (5ug/ml) and I don't know why. I'm suspecting that it is some kind of cell clumping phenomenon? When I re-streak some of these "false transformants", they don't come up. Please help!
-Meltdown-
I've never worked with bacillus but can you increase your kan concentration? With E. coli our lab uses kan50.
-microgirl-
Meltdown on May 8 2009, 12:02 PM said:
Hi there!
I've been trying to transform Bacillus subtilis with a plasmid carrying kanamycin resistance to knock out a gene. I use Spizzizen's protocol. My problem is, my "cells only" control (no plasmid added) keeps growing on the TBAB+kan plates (5ug/ml) and I don't know why. I'm suspecting that it is some kind of cell clumping phenomenon? When I re-streak some of these "false transformants", they don't come up. Please help!
I've been trying to transform Bacillus subtilis with a plasmid carrying kanamycin resistance to knock out a gene. I use Spizzizen's protocol. My problem is, my "cells only" control (no plasmid added) keeps growing on the TBAB+kan plates (5ug/ml) and I don't know why. I'm suspecting that it is some kind of cell clumping phenomenon? When I re-streak some of these "false transformants", they don't come up. Please help!
Firstly, you must sure that whether kanamycin is to lose efficacy.
Then, increase kanamycin concentration.
-daweiyjao-
We routinely use kan at 30 ug/ml, but have gone as high as 50 ug/ml. Be sure to allow a non-selective expression period before plating your transformants.
-HomeBrew-