posted 3 months ago

Has anyone ever optimized/troubleshot a NASBA RNA amp before?

I have a mentor whose company is struggling to optimize a NASBA reaction. The reaction does amplify the target, but it takes over an hour, and the target yield is low, with lots of non-specific amp.

If anyone has any ideas on how to optimize, this would be greatly appreciated!

Guy Rohkin3 months ago

although haven't done much of these I can give my two cents on amplification experiences in general (from working with RNA derived libraries/genomic based applications).

  1. firstly there is just the basics, MgCl, pH, other buffers (although to be honest I didn't find these to be the culprits usually)
  2. primer design -- the non-specific amplification and low yield would indicate that to me. Really double, triple, quadruple check that...that was usually the problem.
  3. RNA quality? it could be degraded, but I assume that was checked at the onset
  4. enzyme -> expiration date...I know it's obvious but I have made that mistake too~
  5. blast it with more primers, more input, more enzyme
REBECCA VAUGHT3 months ago

This is awesome @Guy, thank you! I will pass along.

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