| | Re: Troubleshooting for real-time RT-PCR
I have posted a reply to luisillo, it said moderator's approval is required.
Anyway I think I will write again, what I was trying to say was that I was confused with the result as if really contamination happened, both targeted gene sample and its respective no template control would have same two peaks in the melt curve analysis if I mixed the reagents together (1 tube being transferred equally to 4 tubes, cDNA added separately), am I right to say that? Now the no template control had a distinct product judged from the melt curve performed (for Bax and Bcl-2) except for GAPDH which clearly has an amplification exactly the same as the targeted gene sample. Is that really a sign of contamination?
Anyhow, I would check the real-time PCR product I had on agarose gel.
For luisillo: I don't quite get you about the reagent testing. Do you mean testing my real-time PCR mix by running it on conventional PCR or testing my real-time PCR mix by running real-time PCR and afterwards running gel to see the bands?
For butters: the mix is purchased commercially, it is sensifast SYBR hi-rox kit from bioline. From what I mentioned that I followed the published paper was the primer sequence. And I tried the cycling method also, but it did not work. Everytime I run the experiment, at least 2 genes' NTC would have amplification.
I am actually using strips, just that in order to avoid contamination, I cut the strips into 4-tubes strips. Once I finished mixing for one gene sample, I would straightaway close the tube with the lid. The 8-tube strips are supposed to be sterile I guess??
Do correct me if I said or did something wrong. Pardon me as this is my first time doing real-time PCR. Thanks.