Hot answers tagged reverse-transcription
I see big fuzzy bands around 100bp as well. They're most likely RNA contamination. To get rid of them, digest your RT-PCR products with RNAse-H. But if you just need to visualize your band of interest, and the fuzzy bands aren't getting in the way, it shouldn't be a problem. I usually input anywhere from 1-2 ug of RNA into my RT-PCR reaction using the ...
There is no single "ideal amount" of RNA. I would suggest you to do a titration curve to determine the best amount for your specific assay. The band at 100 bp could be a non-specific amplification. You could try to increase slightly the annealing temp to see if that removes (or reduces) the lower band. Alternatively, you can consider a different primer set ...
The best way for scaling up these kinds of reactions is to set up many reactions. Prepare a mastermix for lets say 20 reactions. You can pool them together, precipitate the RNA and dissolve to appropriate concentration in nuclease free water. Also, use gene specific primer instead of oligo-dT and don't add poly-A tails (poly-A does not affect the in-vitro ...
RT-PCR should be able to get you up to ~10kb. If you are finding that it is not working, then you can by long-range RT-PCR kits commercially (look at Stratagene and Qiagen, for example). The Qiagen can handle up to 12.5 kb in a two-step RT-PCR system.
Nanodrop may be fine for the volumes but as you rightly guessed it is not so easy to distinguish DNA from RNA (and primers). Qubit (invitrogen) is a sensitive, dye based quantification assay. You can use that for estimating cDNA yield. The kit contains different dyes for DNA, RNA and protein estimation.
You have to design your primers properly. Usually, in real-time PCR, you don't choose a very long product. Ideal product size is 150-300. Next, see what your NRTI is analogous to. For e.g. if I am using AZT (Azathymidie), I would place my reverse primer at or after the last T. There are alternate techniques as well. You can use primer extension ...
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