cloning dsr

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.

Mike


>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.
>
>kind regards,
>Andrew.


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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