PCR from phage suspension

"Alexander Kraev" bckraev at aeolus.ethz.ch bckraev at wawona
Fri Apr 7 03:44:59 EST 1995


In article <3m165r$5k0 at synapse.bms.com>, Steven Goldberg <goldberg at bms.com> writes:
>I have a number of cDNA clones in gt11 that I want to screen using
>PCR.  Single plaques were picked and diluted into 0.1 ml SM buffer.
>Assuming the SM buffer doesn't interfere with PCR, how much of my
>phage suspension should I use in a standard 0.1 ml reaction?  Or are 
>there any alternative protocols that someone can suggest?
>
We routinely screen the phages, suspended in SM, in the following way:
1. 1 ul of phage is added to 9 ul of water, containing 5-10 pmol of each primer
and overlayered with 30 ul of oil. The mix is placed in the cycler and heated
at 95 C for 5 min.
2.2X Taq mix is prepared, containing buffer, polymerase and nucleotides.
At the end of the 5-min step the cycler is paused and 10 ul of 2XTaq mix is
added to each tube. The program is continued for 35 cycles. 8 ul are analysed
on a gel.
Be sure to include a genomic DNA control with your samples, since very
frequently there are bands of different size, apparently generated from lambda
or E.coli DNA. This worked so far for more than 30 different primer pairs.
Boehringer now sells "PCR Master" mix, which fits very well into this protocol.
Cheers,

*************************************************************************
Alexander Kraev, Ph.D.                 Internet: bckraev at aeolus.ethz.ch
Lab. of Biochemistry III               Phone: 0041-1-632-31-47
Swiss Federal Institute of Technology  FAX:   0041-1-632-12-13
Universitaetstr.16
CH-8092 Zurich
"Some ideas are obscure not because they are complex, but because they 
 are excluded from our circle of comprehension" - Kozma Prutkov



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