paranormal qpcr amplification activity
Posted 20 May 2010 - 03:47 AM
I have been reading your discussions for quite a long time, and they always helped me. Today I decided to post my own problems, and I would be very happy if any of you could help, me because I am getting desperated!!
as a summary: I have 3 genes to investigate (1reference and 2 targets). I did my standard curves, check amplification efficiency and so on... Now I am checking my samples (which are a mixture of RNA extracted from larvae infected with bacteria; my targets genes are specific for the bacteria), since there is no way to separate it to get a clean bacterial RNA. I do specific retrotranscription (with a mixture of all my primers, since my sample amount is limited) and perform my qPCR, and all my problems are arising...
When I check my melting curves sometimes I get different peaks and sometimes is everything perfect, I always perform the same protocol, nevertheless sometimes I see a perfect peak at the right melting temperature, and sometimes it is a great chaos. What could be affecting my results?
Other thing taht really frustrates me, is that some times my melting temeprature is changing among replicates... although i pipetted it in the same way, and normally my replicates dont have a great variation in Ct.
Can somebody help me? or give some kind of advice?
thank you very much in advance!!
Posted 20 May 2010 - 04:39 AM
Variation of PCR product melting temperature deduced from dissociation curve between + / - 1°C is normal as it is affected by many factors which can change between runs (for example salts in template).
Posted 20 May 2010 - 05:09 AM
I did a temperature gradient for my different primers, and I am using the one that was quite efficient for all my amplicons, since I want to run them together. I will low the primer concetration (in which range should be appropiate?)
thank you again!