Dr I.S. Viney
iviney at hgmp.mrc.ac.uk
Wed Sep 11 08:14:24 EST 1996
We use the Promega T-vector and have always got our PCR product cloned.
We just check that the product is at least detectable in an agarose gel
after Geneclean. If no transformants are obtained (result in about one
third of ligations) then the amount of vector DNA is doubled and the
amount of insert DNA is increased (usually the rest of the geneclean
yield), this has (in our hands) always resulted in transformants. Some
blue (lacZ+) colonies always appear but so far the majority of white
colonies always contain our clone. Of course we then sequence our clones
to ensure the absence of PCR-generated mutations. I have no affliation
with Promega but I am satisfied with this product, especially as it has
worked without me working overtime on quantifying the DNA and hasn't
required lengthy purifications.
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