PCR of phage library
Anton Scott Goustin
asg at cmb.biosci.wayne.edu
Thu Sep 14 18:28:41 EST 1995
Steven Goldberg <goldberg at bms.com> wrote:
>I am trying to clone a gene from a porcine cDNA library in gt10 using
>PCR. The insert is ca. 1700 bp and its DNA sequence is known. So far I
>have been able to amplify fragments of 500 and 600 bp from both ends of
>the cDNA using an aliquot of the denatured phage library as template,
>which is exactly as predicted. However, I can't get the full-length
>gene to amplify, although the primers representing its NH2 and COOH
>termini are the same used in the successful experiments described above.
>What sort of modifications might be useful to obtain the longer PCR
>product I'm looking for?
>Thanks for any ideas.
Clontech and New England Biolabs both sell specific 24-mer primers which anneal adjacent to the Eco RI site in lambda gt10 and point toward the Eco RI site. Buy them or synthesize your own after the catalog. Just do PCR on the phage DNA and you will get 1.7 kb. You can even just do PCR on a plaque, if you are too lazy to purify the DNA. It works just as well. I have retrieved inserts as large as 2.5 kb no problem. The inserts will have Eco RI sites near their ends, too, so you can check an aliquot of the PCR reaction (just add EcoRI directly to 5 µl of the raw PCR reaction) for internal Eco RI sites. If the insert is still 1.7 kb, then treat the rest of the PCR product with Eco RI and clone into a plasmid at its unique Eco RI site. Don't worry about the fidelity of Taq. It is good. If you don't believe me, produce 5 plasmid subclones of your lambda clone via PCR popout and sequence the 5 plasmid clones You will probably find them all to be the same. Taq is not as error-prone as you think. But DO sequence any clone that you do subclone. It's only 1.7 kb and you don't want to get caught months later with a bad clone!
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