Insertion into an expression vector.
wlauzon at uottawa.ca
Thu Mar 1 11:01:47 EST 2001
We have a BamHI/NotI cDNA fragment that encodes a mature transcript without
a start codon. We wish to insert it into an expression vector. Because of
the lack of a ATG and the intervening S&D site, we have constructed oligo
linkers to fill in the necessary sequence. oligo 1 is a 29-mer that has the
conscensus sequence for BglII(restriction site on the vector) the S&D site
and an 8pb sequence complementary to the end of oligo2. Oligo2 has the 8bp
complementary sequence for oligo 1, a start codon, and BamHI complementary
sequence (insert restriction site).
The question is, what is the best enzyme to use, to fill in the single
stranded sections of the construct? We are looking at Klenow and T4
polymerase. Are there drawbacks to either one? Are there others more
appropriate to our needs?
Thanks in advance,
Wallace Lauzon, PhD
Dept. Biochemistry, Microbiology, and Immunology
Faculty of Medicine
University of Ottawa
451 Smyth Rd
Phone: (613) 562-5800 x8244
FAX: (613) 562-5452
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