I have thus used superscript II and IV :/ I denature RNA at 65 degrees, I wonder if I can increase that to a temperature at which DNA is usually denatured at (around 94 degrees) without compromising RNA degrading... My target amplicon is just below 500 bp.
As far as I remember, superscript IV troubleshooting suggests prolonged (
- Thanks Asta, I did the SSIV reaction as suggested at 55 degrees and still nothing from PCR. I am very confident in my PCR because I have three positive controls of other genes from same RNA sample that work really well, I think because their transcript is much lower in GC content; I have sequenced one of my positive control extracts to ensure it's a real positive control and was 100% correct as predicted. And I know the gene I am trying to amplify is definitely more abundant than the other two genes I use for positive control. I also tried gene specific primer for that, and got a smeary band on my EtBr. I can see something very faint around the weight of my amplicon, but its definitely not pure and I don't think trying to cut around that gel will be useful especially since so much is lost in column purification.
Any MMLV enzyme can use for RT. You can use any hot start tax pol. for >%65 GC content. In addition to, you can increase the denaturation time for PCR. It works most probably.
So, because the controls are lower in GC content, you are getting the PCR fragment and it shows that the system works for that %GC.
Have you thought about PCR high-GC control?
I did some back-thought thinking :)
In your case, I'd make PCR control reaction with >65% GC DNA fragment.
Then, use random hexamers + 55°C + 1h for cDNA synthesis. Then use varied amount of cDNA for PCR. Be wary, though, the hexamers might result in additional short PCR fragments.
Hope this helps.
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