Michael L. Sullivan
mlsulliv at facstaff.wisc.edu
Mon Nov 6 10:20:38 EST 2000
Three methods I've used with success are to:
1) design your primers with restriction sites. Make sure to add 4-6 extra
bases so that restriction sites are not at the very end of the amplified
product. This makes the cloning routine.
2) I've made my own T-ended cloning vector using a protocol in the Red Book
(Current Protocols in Molecular Biology). Basically, use any vector (one
with color selection is a good choice) cut with a restriciton enzyme that
leave a blunt cut (like EcoRV) and incubate with Taq Polymerase in the
presense of dTTP only. This adds a T compatible with the A so often
introduced on your PCR product.
3) Blunt the PCR product ends with something like T4 DNA polymerase and
clone the resulting blunted product into a blunt-cut vector. Again, color
selection can be helpful here.
Hope this is helpful.
>I'm a PhD student working in microbial ecology. For quite some months I've
>been trying to clone the dissimilatory sulfite reductase (dsr) gene from
>environmental samples. As the primers are degenerate, I have multiple bands
>and preparation for cloning requires gel excision and purification. Have
>tried pGEM-T, pCR script, pCRII, pCR-TOPO, pCR-TOPO-XL.... as you can all
>imagine, a lot of money and a lot of sweat (and tears!) have gone into this
>process. Any experience/advice greatly appreciated.
Michael L. Sullivan, Ph.D
U.S. Dairy Forage Research Center
1925 Linden Drive West
Madison WI, 53706
(608) 264-5144 Phone
(608) 264-5147 Fax
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