Does anybody have or know a protocol to understand how to optimize RNA/random hexames/temperature in ds cDNA synthesis reaction to get a precise fragment size?
You will never get a precise fragment size; the RT reaction will always yield a population of fragments of varying lenghts. I don't think you can predict average fragment size from the get go, instead you have to optimize your experiment by making trial runs varying different parameters until you get the desired result. In general:
- The higher the quality of your input RNA, the longer the resulting cDNA in average.
- Using oligo dT will result in larger first strand sizes (but the 5' of all RNAs will be underepresented).
- Using a modified RT (say, SuperScript III) instead of native MMLV or AMV RT will also yield longer cDNA.
- If bound to use random hexamers, decreasing the amount of hexamers in the reaction will decrease yield, but will increase average cDNA size. The same thing will happen if you increase the temperature (but then you have to play around the limitations of your reverse transcriptase).
Hope it helps.
Thank you!
Will it be ok if I add random hexamers just once, in the first strand synthesis right?
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