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6

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 ...


4

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 ...


2

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.


1

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.


1

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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