Insertion into an expression vector.

Dr. Hiranya S. Roychowdhury hroychow at nmsu.edu
Thu Mar 1 21:14:42 EST 2001


Both enzymes work fine. T4 is also useful in chewing back 3 prime
protrusion (3'-5' exonuclease activity) which the Klenow fragment does not
do. 

This should help you decide on the enzyme.



At 11:01 AM 3/1/01 -0500, Wallace Lauzon wrote:
>Hi,
>
>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,
>
>Wally
>
>--
>Wallace Lauzon, PhD
>Dept. Biochemistry, Microbiology, and Immunology
>Faculty of Medicine
>University of Ottawa
>451 Smyth Rd
>Ottawa, Ontario
>CANADA
>K1H 8M5
>Phone: (613) 562-5800 x8244
>FAX: (613) 562-5452
>
>
>
>
Dr. Hiranya Sankar Roychowdhury
College Asst. Prof.
Molecular Biology,
Dept. of Chemistry & Biochemistry	
Box 30001 - 3MLS
New Mexico State University
Las Cruces, NM 88003

Lab: (505) 646 4722
Office: (505) 646 8256
hroychow at nmsu.edu


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