My negative control in each PCR reaction/ thermocycling was based on not adding cDNA to the reaction. But in the same PCR, I always used the GAPDH housekeeping gene primer. Is it right to use it as a positive control? It was delivered with the kit and I don't know the primer sequence?
It is always good to have a negative control to show contamination problems which often occur with PCR. GAPDH is widely expressed and it will give you confidence that your cDNA is good quality when GAPDH amplifies. Otherwise if your gene does not amplify you do not know if your cdna is low quality and nothing will amplify or if GAPDH works and your gene fails then it is likely to be a good result but negative for your gene rather than just "it did not work"
It depends on your polymerase system, you must refer to the manufacture guid. basically positive control is an known pattern (Ct) tell you that every PCR components especially the cDNA quality and quantity are working well. It’s better to start an initial experiment contains only three samples positive, negative (to make sure that there is no contamination) and housekeeping gene. The GAPDH in your kit is a universal, you have to use an endogenous housekeeping gene of your organism such as Actin, Tubulin or ubiquitin.
Finally, the best is to use the housekeeping gene primer with annealing temperature similar to your GOI.
I may have misunderstood part of your question Ismail Zeb . GAPDH is ideal as a positive control for amplifying from a cDNA template but primers for use in RTPCR are designed to cross intron/exon boundaries so that genomic DNA does not amplify or produces a very large amplimer so many primer sets designed for RTPCR will not be suitable as a positive control for amplification from genomic DNA
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