Heated-lid - (Feb/14/2012 )
Just a quick question. Today I ran a PCR and forgot to lower the heated lid so there was no contact between it and the tubes. The program ran through, however. There was no loss of volume but I'm worried the reaction didn't proceed due to condensation. I'm using this product as template for a second round and there is never enough product to visualise after only the first round. Is there a chance that the reaction was successful? I ran 35 cycles so I'm thinking about sticking them back for 5 cycles with the heated lid lowered properly. Do you think this would help?
Thanks so much
can't you run on a gel really? not even 2 ul? I think it should be fine. When PCR was done in water bath it was very popular to use oil on top of the reaction. I think this habit has been continued until now. but these days we run in 250 ul tubes. the block already covers most of the microtube.
I could run it on a gel but I wouldn't get the faintest of bands. I'm amplifying from single cells so the amount of DNA after just one PCR is very little. I didn't put oil because my cycler has a heated lid, I just forgot to lower it.
Never did this, so I'm not sure, but IMO it depends on the PCR reaction characteristics:
A stable PCR that usually works almost always, independent from concentrations, pipetting errors, DNA amounts etc, has a much higher chance to work in this situation too. The more volume the reaction has the more likely the reaction worked (i.e. 10 microL less likely than 25 microL).
Anyway I'd also would check with a gel, even if it's a faint band, it's an indication. And finally it depends on time and costs, i.e. if the second round is time consuming and expensive then I'd start again just to be sure not to waste too much time and money.