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colony PCR after transformation

bacteria DNA transformation REcombination colony PCR

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#1 phage



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Posted 22 July 2013 - 10:17 AM

Hi All,

I have encountered a problem that's hard to explain. I would really appreciate if someone could shed light on what's going on.

I am trying to see whether I can delete all 4 genes in a bacteria strain. Currently, this strain already has 3 of them deleted (replaced by 3 antibiotic cassettes), but I was unable to delete the No.4 gene using a PCR product that contains an antibiotic cassette flanked by No.4 gene flanking regions.

Therefore, I am using an alternative strategy: delete this No.4 gene by transforming the 3 genes-deleted strain with a chromosomal DNA. The DNA has the No.4 gene replaced by antibiotic cassette. However, the chromosomal DNA also contains 3 wild-type genes that could be recombined back into the accepting strain. My goal is to see whether all 4 genes can be deleted, or one of the genes is required for bacteria viability.

I was able to get No.4 gene deleted (verified by colony PCR). The colony PCR told me that I have the antibiotic cassette flanked by No.4 gene flanking regions. I was also able to verify that gene 1 and gene 3 are still in "deleted" state. However, I could not get the PCR product of gene 2 deletion, while my positive control (the PCR for gene 2 deletion on the 3 genes-deleted strain) worked beautifully. I did two more PCRs, which is to see if I can get the amplicons of upstream flank + antibiotic cassette and antibiotic cassette + downstream cassette (see figure below). These two PCRs worked beautifully, and the PCR size are consistent with what I predicted.

My question is, is there an explanation for this inconsistency? You can have PCRs of two DNA sequences work, but not of three DNA sequences.

Thanks to all.


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#2 bob1


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Posted 23 July 2013 - 01:55 AM

One possibility is that gene 2 is located in close proximity to gene 4 and was disrupted by the addition of the resistance cassette/ko procedure.

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