Cloning IPCR Products

Fernando Cardoso fernando.cardoso at ineti.pt
Fri Aug 31 12:13:24 EST 2001


Hi!
first tray to isolate a new transform bacteria colony from your stock.
second try to gel isolate the 2 bands and use direct PCR sequecing
Fernando


"Stephen Dibley" <stephendibley at ozemail.com.au> wrote in message
news:3B8EFC49.88F4C8A9 at ozemail.com.au...
> 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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