Some info. on PCR products stability w/respect to shipping - (Aug/31/2014 )
Just wanted to share this as it may be relevant to saving money when shipping PCR products for sequencing.
Where we send the company website says they want overnight and in a cooler with dry ice (for crude PCR products in the PCR buffer solution). Theoretically and from a (inadvertent) past experience (involving 2 days at RT), they should be fine, for some time at least (if not having been open) at RT, because they’ve effectively been sterilized + any DNase enzymes degraded by being at 94-95 C.
I also did (gel pic attached) a check involving leaving two tubes at RT for ~48 hrs after they had been briefly opened (and thus potentially contaminated) at the bench in the course of running a gel. No difference was observed between them and the same product that was at -20 C the whole time.
I make this post because it might save some people a considerable amt on shipping to send it w/o cooling and possibly even 2nd day instead of overnight… as it would appear from my experience that virtually no degradation occurs (at least not within a few days).
P.S. Does anybody still do a final cool holding temp. after running a PCR? I would say it’s totally unnecessary (even if left overnight) and I’ve read it will speed up the machine wearing out, due in large part to the potential for water condensing and ruining the electronics.
Your findings are correct... PCRs are usually limiting in one or more of the reagents (usu primers and dNTPs) and the polymerases, while thermostable, still degrade with each cycle, so after a typical PCR, there shouldn't be any further amplification of either contaminants introduced after the reaction, or specific product. I've always shipped my sequencing PCRs just in a regular courier bag and never had any problems.
Surely the last step of my PCR is a cooling step to stop (or slow down) the reaction but at 10 degrees and not at 4. Just to be sure that nothing is happening.
And the enzymes still work after a reaction of 30 cycles since they also work with 35 or 40 cycles, i.e. something is still alive after a normal PCR. And I want a PCR clearly stopped after the cycle number I programmed and not a continuation as it wants even if slowly at 20 degrees.
At worst, the current cycle will complete (which is the purpose of the final extension, in any case). Further amplification cannot take place, since the primers have no chance to re-anneal, since the previous product is not denatured.