small PCR product-->ligation
kucera~gt at glaxo.com
Thu Mar 21 08:36:16 EST 1996
luis <lgbrieba at academ01.mty.itesm.mx> wrote:
> I have a small PCR product (300 base pairs) afther the cut with
>KpnI and AflII, that need to be inserted in a plasmid. Is the first time
>that I do a ligation (T4 DNA ligase) and I have doubts if the size of
>the insert may influence the efficience of the reaction. Could anybody
>give me advices for improve the reaccion?
> Thanks in advance
The method outlined below takes into account the number of pMol ends available for ligation of vector and insert:
Begin by calculating the number of pMol ends per microgram of DNA using the following equation:
2 x 10E6/(660 x bp)=#pMol ends/ug DNA
I typically use a ratio of 1:5 pMol ends (vector:insert) for blunt end ligations, and 1:3 ratio for cohesive ends.
A 300 bp fragment should not be a problem. You will find based on the above equation that you will not need very much insert DNA in the ligation (approximatly 2 fold less insert than vector assuming you are cloning into a 3kb vector, blunt-ended) .
Gary T. Kucera
GlaxoWellcome Research, Inc.
Molecular Biology/Transgenics Group
Five Moore Drive
Research Triangle Park, NC 27709
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