Cloning IPCR Products

Michael Witty mw132 at mole.bio.cam.ac.uk
Fri Aug 31 02:34:46 EST 2001


Dear Stephen, if it was me I would either try repeating the IPCR, or if
that is impossible, subclone your two existing clones to a different
plasmid and then sequence them (to see if you should continue with them.
Regards, Mike.

On Fri, 31 Aug 2001, Stephen Dibley wrote:

> Does anyone have any experience with cloning inverse PCR products? I
> generated two fragments via inverse PCR and cloned them into the cloning
> vector pDrive (Qiagen). Digestion analysis of these plasmids shows that
> they contain the correct size insert, but PCR analysis on these plasmids
> always produces multiple bands. This occurs with both sequencing primers
> (SP6 and T7) and insert specific primers. I have increased the annealing
> temperature as still recovered two bands. This problem is also stopping
> my from sequencing the insert. Any suggestions on what to do would be
> appreciated.
>
> Thanks in advance,
>
> Stephen
>
>




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