PCR on Lambda

Mick Jones mjones at rpms.ac.uk
Mon Nov 13 05:39:16 EST 1995


Dear Carl

We have sequenced cDNA inserts in lambda gt10 and gt11 phage libraries 
by direct PCR amplification from picked plaques.  Insert sizes 500 bp up 
to 2 kbp reliably amplified.  All we did was to toothpick a plaque into 
the PCR mix.  We analysed over 1,000 cDNA sequences.  The success rate 
for amplification was about 60%.

With hindsight I would do it as cescribed below:

Pick a plaque into 20 ul of PCR buffer using a plastic/steel toothpick 
(just in case any inhibitors in wooden toothpicks/cocktail sticks).  You 
could then twirl the toothpick in 20 ul lambda phage storage buffer as a 
back-up.

Then take 10 ul into a PCR reaction.

Good luck

Mick
_____________________________________________________________________
Mick Jones                      Tel: 081-740-3328 (+44-81-740-3328)
Department of Virology          FAX: 081-743-8331 (+44-81-743-8331)
RPMS,  Du Cane Road              Email: mjones at rpms.ac.uk 
London,  W12 0NN,  UK          URL: 
http://www.rpms.ac.uk/virology/virolhome.html
***********************************************************
"Smoke me a kipper, I'll be back for breakfast."   Ace Rimmer (Red 
Dwarf)
"Crackin' toast, Gromit."    Wallace (The Wrong Trousers)
_____________________________________________________________________





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