Problem with repeatability of the standard curve - (Dec/13/2013 )
I´m a newbie in these PCR issues and today I have a problem that I hope someone could answer.
This week I ran my first qPCR plate, used 5 serial diluted standards and I got nice results (slope= -3,341; R2=0,999; Efficiency = 99,21%). So I was very happy..... But today I had to prepare another plate, and I used the same standard dilutions, same pippettes, the same kind of tips, the same mix,... and I got a slope= -4,033, R2 = 0,986 and efficiency of 77,86% . All the reagents (standard, probes, primers, mix,...) are supplied by the commercial kit that I´m using.
I know that these two standard curves are not comparable, since they are too different and I´ll have to repeat the second plate because the curve is very bad and the results of quantification will not be able to compare between both plates....
but my question is: where can the problem be? Why, if I´m using a perfectly standarized commercial kit (presumably), do I have so much desviation?
Sorry for these noob questions, for my English and thank you in advance for your answers.
How did you store the kit - freeze/thaw cycles will damage most DNA quite efficiently. Templates and the like would be best aliquotted for single use.
I assume that you forgot to mix your standards on the second plate before pipetting. Mix really good after thawing (vortex each tube for sereveral seconds, not just pippeting up and down) and try your std curve again.
Usually, std curves are reproducible and it is not necessary to run them on every plate.
thank you everyone for your answers!
I don´t think that the freezing/thawing was the problem, because the kit was storaged at -20C as the manufacturer instructions say and this case was the second use of the kit. All samples and standards were in ice during the preparation of both plates, too.
In my opinion it could be more related maybe with a bad vortexing of the standards, but I´m pretty sure about I vortexed all samples that day (and after that I always centrifuge the tubes in order to not have any drop in the lids)... I will repeat the second plate and I expect to obtain better results.
thank again both of you for your time answering me,