What is the best way to handle PCR fragments without damaging them?

[rockind78]

I ran a PCR last week but did not get around to running the AGE until this week, so the PCR product sat for a roughly a week at -4*C. What are the odds that all my DNA is at the bottom or stuck to the sides? A friend of mine just suggested this to me, or I would have vortexed it before running the second AGE. Any thoughts?

 

[iansmith]

Sedimentation migth probably happenned. It will not hurt to shake the reaction but vortexing can damage DNA if it is too long (more than 10 sec.). So a short pulse (3-4 sec.) should put everything back into solution.

In the other hand, the DNA migth have started to degrade. A week in the frigde is not that long and the degradation should be minor. It always best to store PCR reaction in the freezer (-20C).

 

[rockind78]

Originally posted by iansmith
It always best to store PCR reaction in the freezer (-20C).

I biochem professors would agree with that. Oddly enough, my molecular biology professor prefers the fridge. Thanks for the input though. That is just what I suspected.

 

[Monique]

Do PCR fragments really settle?? I actually don't think so. I don't think so either that vortexing will damage your PCR fragments, how long are they? There really only is shear stress with genomic DNA, where flicking a tube with your fingers is adviced.

 

[iansmith]

Originally posted by Monique
Do PCR fragments really settle??

Without any movement it will but very slowly.

Originally posted by Monique
I don't think so either that vortexing will damage your PCR fragments, how long are they? There really only is shear stress with genomic DNA, where flicking a tube with your fingers is adviced.

When we order primers, the instruction to reconstitute them says to limit the vortexing to 15 secondes. The kit we use for genomic extraction also suggeste to vortex but to limit to 15 sec because shearing will start to occur.