PCR using lambda gt11 primers
jfhess at ucdavis.edu
Thu Mar 9 16:31:12 EST 1995
Provide more info for us. I have amplified inserts from lambda phage.
Since gt11 should be used with cDNA libraries, the inserts should be
smaller than genomic inserts. This means you should have success with
PCR. Make sure you have the right oligos for the phage and the cloning
site. Titer the phage so you know how many you're adding to the pcr
reaction. Keep addition of phage (in SM buffer, right?) to a minimum
<10% of PCR reaction. I would start with something like 94, 30sec, 55,
30 sec, 72 1minute.
30 cycles SHOULD be ok, 35 definitely and if you try more, it's not
working for you.
If you have primers to the insert, do an insert specific primer with each
possible end of the lambda phage cloning site. this will orient the
clone as well as cut down on the size of the potential PCR product (just
in case this is a problem).
In the last week, I have amplified 2 charon 21a inserts of human genomic
dna. One was 2 kb, one was 3 kb. However, the full insert (6 kb by
cloning) was not PCRable. I had to clone the insert by digestion w
Good luck, email me directly if you have more questions.
John Hess, PhD Phone me 916 752 8420
Dept of Human Anatomy FAX me 916 752 8520
University of Calif Email me jfhess at ucdavis.edu
Davis, CA or leave me alone, your choice.
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