Can you repeat a thermocycle? - (May/28/2014 )

I ran a PCR and it was not successful, as judged by viewing on a gel, no bands. So, if I placed it in the fridge, could I lower the anealing temperature of the thermal cycle and just re-run it the next day? With the same tube, same reagents and everything from the previous day? Would this work? I suppose I would be using up more sample to view it on another gel, 25uL pcr, used 5uL for gel, will have to use another 5 to test again, so I'll be left with 15uL. The template DNA is precious, and he primers have worked previously, albiet they produced weak bands, but I'm hoping I could just throw the same rxn tube back in the thermal cycler at a lower annealing temperature and it will magically work the second time. Any idea?

Short answer - no.

You would need to add more polymerase (despite heat resistance, still gets damaged by the cycling) and potentially the template could have been degraded if you are using a proof-reading polymerase.  However, dilution of your template may actually improve the results.

Hi,

You must first optimize the pcr with a positive control and then proceed for the precious DNA, the amount of DNA is very important, may be your template needs to dilution. 

thanks for the feedback guys. i appreciate it.