PCR from phage library

Thu Apr 27 17:17:50 EST 1995

>> Steven Goldberg <goldberg at bms.com> wrote:
>> Would it be possible to PCR out a ca. 2000 bp fragment from a lambda
>> gt11 cDNA library.  The library is from pig liver mRNA and there are
>> ca. 10 to the 9th phage per ml.  Unfortunately I don't have any cDNA
>> as a control since the library was purchased commercially; however,
>> the sequence of my desired gene is known so I can synthesize unique
>> primers to it.

>> Any ideas on how I might go about this would be appreciated.

>> Thanks.

>> Steve Goldberg

	It may be best to dilute 2-5 ul of the phage soln to 50 ul with H2O,
add 1 ul of chloroform, heat to 95 oC for 5 min,put on ice, and microcentrifuge
at top speed. Use 2-20 ul of sup as template in 50 ul PCR. However,some people
directly use the phage suspension in their PCR. I have done lots of trials
for a similar problem, but finally had much better results when I used
the original cDNA (or mRNA for RT-PCR) as a template instead of phage.
Hope this helps,

Gerard Drewes
MPG Struct. Biol. Unit
Hamburg, Germany                                            

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