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Mick Jones mjones at rpms.ac.uk
Wed Dec 20 12:17:00 EST 1995


Hi netters

I guess this may be an item already discussed on the net, but here goes.

We are doing some RT-PCR, and have generated the correct sized bands (on agarose gel anyway).  We 
used random hexamers to prime the RT reaction, and then used gene specific primers for the PCR 
step.

What I would like to do is prepare more of the frgament by re-PCR amplification with the same 
primers (we do not possess any nested primers), as there isn't very much product with the first 
PCR.

I tired diluting 1 ul of first PCR into 200 ul water, and used 1 and 10 ul of this as the template 
for a second PCR using the same original primers.  Result no joy at all.

So,

Has anybody a tried and trusted method of taking an aliquot of a first PCR and then carrying out a 
successful second PCR to give nice clean high yield of desired product or know a man who can?

Urgent replies required as it is getting close to Christmas, and I would like to be home with 
kids, warm slippers on feet, real fire burning, glass of scotchin one hand, cuddling up to the 
nearest and dearest....

You get the picture.

Seasonal greetings from a cold and damp London, UK, to ONE and ALL around the world.

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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