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