HELP with PCR

Jörn Lewin joern.lewin at epigenomics.com
Tue Oct 23 04:02:49 EST 2001


Hi Krzysztof,

did you try different MgCl2 concentrations in your buffer? I worked some time with denaturated
primers and had to find PCR-optima several times. I think there are several important parameters
beyond the PCR-program (temperature/time). Maybe it is nice to try different kits and other expensive
stuff or even destroy the lab (which might be not too useful but at least very expensive). But I
recommend to go back to some basics and vary the following parameters:

- MgCl2 (as mentioned)   0.5 , 1.0 , 2.0 , 3.0 mM
- Primer concentration   50 , 100 pmol
- maybe Template concentration

If you did not vary those parameters yet, give it a try.

Topic hotstart: You won't need a pre-heated block but a pre-denaturated sample. Assemble your samples
without TAQ, put them in the block and denaturate for 3 - 4 min at 95° C. Then quickly add the TAQ
into the tubes in the block (in the one more minute at 94 °C at the beginning of the first cycle,
calculate more time at that temperature if you need it for your performance).

If everything fails, try to find an old indian who teaches you the PCR-dance. Good luck
--
Joern Lewin --- Bioinformatics R&D
Epigenomics AG, Kleine Praesidentenstrasse 1, 10178 Berlin, Germany
www.epigenomics.com
Tel.:  +49-30-24345-355





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