reamplification techniqes - (May/28/2008 )
I amplified several DNA extracts and some of the samples showed very weak bands in the gel.
So, to play around, I decided to take one microliter of these PCR products and reamplify them with the same set of primers and conditions.
They showed up beautifully in the second amplification but there was also an extra band.
I've been playing with different parameters such as annealing temperature and cycle number. But I continue to get the extra band. Otherwise, the gels are very clean with no smears.
Does reamplifying PCR product always produce extra bands or is there a way to get rid of it??
You could try running your first PCR on a gel (run it long to get the most separation between your band of interest and any other secondary products), cut the band out of the gel, purify the DNA from the agarose (I usually just put the gel in the top of a filter tip, put the tip in a microcentrifuge tube, and spin it 30 sec, then use the liquid that ends up in the tube), perhaps make a dilution series of the DNA, and reamplify. You could also try the reamplification with nested primers.
Thanks! I think I'll try the dilution technique.
I usually use nested primers for reamplifying samples that produce good bands. This time I was just reamplifying with the same set of primers to see if I could get very faint bands to produce better bands.