I'm trying to knock out a gene in E. coli using Lambda Red Recombination gene knockout method. I am quite sure I followed the entire protocol carefully, however, when I tried to confirm the deletion using PCR, I faced a very striking situation. I used a set of primers designed to amplify the target gene and was expecting to see a band corresponding to the length of antibiotic resistant cassette (in this case chloramphenicol). But what I saw there was two bands. Sequencing of the gel extracted bands showed that the top band on the gel is indeed the chloramphenicol resistant gene but the lower band is the target gene. I was wondering how is it possible to have both target gene still present as well as the CAT gene?
I chose several different colonies from chloramphenicol plates and faced the same situation with all of them. Any suggestion or idea how it could happen? You might say the CAT cassette might have been integrated into another random area of chromosome but why do I get PCR products of both target and resistant cassette together using the same set of primers?
What was the sequencing data telling you, are you still have the whole gene? You may check the molecular size of both, the target gene and CAT gene is it still the same! When you make sure that both genes are ok, you may got a foreign copy of the target gene (wild-type). Also, you have to pat attention to heterozygous and homozygous KOs issue.
Best Luck
is your negative template control completely clean or is pcr contamination of the target gene from previous setup amplifications a possibility?
@ Ayoob Obaid Alfalahi,
Should have no homozygous and heterozygous knock-out issue here, because it is E. coli (a prokaryote).
@ Ali Kermani,
1. It should be good if you can co-attach a map (see similar experiment in the attachment) and indicate your primers on the map for us to see and discuss. Was your primer design the same as that in the attached figure?
2. I have two thoughts about the results you got: (1) you might get a non-homogenous E. coli population (some cells with knockout, some don't) [but not sure how this could happen], (2) Can some of the successful knockout cells reverse back (also through homologus recombination) if the lamda recombinase enzyme (and a proper substrate) is present consistantly??
Ali Kermani Any luck yet with this problem? I am currently having a similar issue. Please help with an useful information. Thank you.
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