cloning: deletion in a primer

ktorres at edgebio.com ktorres at edgebio.com
Wed Jan 15 09:48:37 EST 2003


Dear Krys,

1.  I don't know about Sure2 competent cells, but would you be interested in a supercompetent BL21 or its derivative for recombinant proteins?
2.  Do you use the Gateway system?
3.  Can you recommend any other Gateway users who could be contacted (including yourself) for an exciting product release or supercompetent cells?

Karen



Krys wrote:

> Hi
> Recently, I cloned a 1000 pb gene in a pET25b+ plasmid. This
> construction was transformed in DH5alpha. The insert was then
> sequenced and shown a single nucleotide deletion in the forward primer
> (It's not a primer problem cause it has been used for another reaction
> without problem).
> To repare this deletion, I used the Quick change mutagenesis kit (from
> Stratagene) and transformed DH5alpha and XL1blue with the mix.
> After mutagenesis: Insert sequences from DH5alpha show the same single
> nucleotide deletion while insert sequences from XL1b show a single
> nucleotide deletion 7 nd after the first (corresponding to the
> mutagenesis primers).
> To summerize: It's not a PCR or sequencing problem; certainly a
> recombination from the host bacteria.
> Questions I submit are:
> 1. What sort of mechanisms bacteria use to delete one nucleotide? Why
> only one and not the same between two different strains (but in the
> same area)?
> 2. This region contains a secondary structure; is it important??? but
> the gene contains at least 50 secondary struct??
> 3. What do you think about Sure2 competent cell in this case?
> Please send me informations or links.
> If you need more information I'm available.
> Thanks a lot
> Christophe





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