Hi everyone !
I'm trying to mutate Bacmid (MultiBac™ - Geneva Biotech) by using lambda red recombination.
after i go through the Red recombination i have to find my Knocked out Cat-SacB Bacmid,
but somehow my SacB does not work in no salt sucrose LB media(6%, 8%,10% tried all..)
I tested SacB in DH5a strain by using plasmid(4.5kb) and it worked,
but my Bacmid(130kb) is in the DH10B strain and the problem happens here.
I can think of some suspicious things, like they, two have different copy number while replicating
and they are in different E.coli strain.
+ I've confirmed that there's no problem with my sacB sequence.
i'm using 315bp SacB promoter [it's shorter than normal sacB promoter gene(446bp)]
Image shows that Red recombination is working well
what seems to be the problem..?
please Help
Thank you for reading :)
I'm a bit confused by the picture. Is what you are seeing the presence of both alleles, the correct KO construct as well as the parent non-KO? If this is the case you may just not have allowed time for complete segregation. You could restreak a few for single colonies and then test some. Especially since the molar ratios of the two are not the same.
Michael J. Benedik Thank you for your reply !!
Since my SacB is not working (cells not be dead that carry sacB gene), i have two types of e.coli mixed in one plate. so i scraped all the colonies from the plate and read particular site by PCR to confirm my Red recombination success.
image shows that i have two construct (KO , non-KO) in one plate.
if the SacB gene has worked, only KO construct would have shown up.
but the problem is sacB not working in bacmid...
Thank you !
it seems more likely that you just have a mixed population, the SacB selection worked but not at 100%. I don’t understand why you scraped all the colonies together, I would just test some individual ones to find the right one. Based on the ratuos of the two bands it seems that most are probably correct
Michael J. Benedik Thanks again sir !
Like you said, first i picked some colonies, one by one to find the right one.
but 100 individual picked was all wrong construct, and i thought it's because of the low efficiency of red recombination. so i tried to find rather my plate has successful recombinant or not. therefore i scraped all of them to check out.
Lucky i somehow found the right recombinant.
leaky working(not 100%) sacB is still problem though...
Thank you for all the replies!!
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