Questions About the Termini of PCR Products

BISHOP at MSRCVAX.BITNET BISHOP at MSRCVAX.BITNET
Thu Sep 26 15:45:00 EST 1991


>From:   IN%"bhjelle at vaxine.unm.EDU"
>I have 2 questions about PCR cloning.

>1. Is it recommended or common now to try to repair the ends of PCR
>products (blunt-ended) before trying to clone them into a vector? I
>understand the ends are often ragged.

>2. In designing a new primer that contains a 5' restriction site, I
>know you should place a few extra bases 5' of the recognition site.
>Are some bases better to use in this function than others?

>Thanks in advance!

>Brian

1.  Taq DNA polymerase adds dATP to the 3' OH of PCR products (Clark, J.M.
    Nucl Acids Res 16:9677-9686, 1988).  This is the basis of commercially
    available (InVitroGen) TA cloning kit. (no affiliation)

2.  We use the non-palindromic sequence 5' GCCGCC which should be long
    enough and has the highest possible tm so as to help keep the ends
    double-stranded.

-David F. Bishop           BISHOP at MSRCVAX.bitnet



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