PCR a 100bp fragment - (Oct/30/2006 )

How do you succesfully PCR up a 100bp fragment and gel purify it (excision from the agarose gel)? I keep getting this smeary "band" which is most likely my primer-dimers. When can you say for sure something on agarose gel is a 100bp fragment and not primer dimers!?

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If the band is smeary, you should play with the annealing temp. The primer dimers should be a little smaller. You can include a control without template (that should be included in every PCR) and see if you have the same band. In that case either you have contamination or primer dimers...

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Using two step PCR program similar to real time PCR:

40~50 cycles of (95C 15S; 60C 1 min), 5pmol each primer for 25ul PCR reaction.
run PCR products on 1.5~2% agrose gel

I have amplify ~60bp fragment by this way and recover it from gel by Qiagene gel purification kit.

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Run a high percentage gel 2-3% agaraose to resolve the different size bands. Also use TBE running buffer and to make the gel.

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