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Unexpected base after deletion mutagenesis

PCR ligase mutagenesis

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#1 crispcrimp

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Posted 20 February 2013 - 07:42 AM

Hello and thanks for reading.

I recently ran into a confusing issue with deletion mutagenesis. The approach I took to the deletion was to amply the entire plasmid, excluding the region to be deleted using 5' phosphorylated primers. PCR was followed by Dpn1 digest and ligation with T4 ligase. I transformed the PCR product into DH5a cells (cloning line) and prepared plasmid from individual colonies.

Sequencing shows that the deletion did work, however there is an additional base where the primers were ligated.

Specifically, the coding strand sequence should have read: ACC.CCT (ligation point is at the period)
and instead I got: ACCCCCT
Where the extra base causes a frameshift in the gene.

I should mention that all 3 colonies that had the deletion also had the extra cytosine.

Does anyone know what is going on here?

--Chris

#2 GNANA

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Posted 20 February 2013 - 08:22 AM

have you double checked your primer sequence??
I would prefer being perfectionist rather than a passionist in Research.

I always had an alternate hypothesis....

#3 crispcrimp

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Posted 21 February 2013 - 09:46 AM

Yes, I got the primers form Invitrogen, and the primer information matches what they should be. It may be possible that the primers are wrong, even though the company says they're OK, but I can't think how to check that.

I should also mention that another lab member has recently had the same problem, with an 'A' inserted where the blunt ends were ligated. At the time he thought that he had accidentally used Taq, which leaves an A-overhang. That can't be my problem though because my inserted base is a 'C'...

#4 GNANA

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Posted 21 February 2013 - 09:59 AM

yeah i thot of that, A overhang could be explained but having extra 'C' is what intruding. Anyways i dnt have an explanation for this may be you redesign a new set of primers obviously the same sequence, and see how it goes.
I would prefer being perfectionist rather than a passionist in Research.

I always had an alternate hypothesis....





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