Hi all, I am trying to extract RNA from 12 well plate and so far the yield obtained from trizol/chloroform protocol is OK.
After that I tried to do DNAase step (using NEB DNAse I) but I found that it does not allow cDNA transcription.
I don't know if it was the inhibition step 70°C x 10min of DNAase activity or smth else.
Do you have any similar experience? if yes, any trick to share with me that can help to do the DNAase step without inhibiting the reaction downstream? Thanks a lot
You could try to repeat the extraction protocol after the DNase treatment, as the protocol is designed to remove any proteins in the sample.
I wanted to avoid that step to not lose material, but thanks for your suggestion :)
Have a look at the ingredients in the buffer supplied with the DNase and compare it to the Reverse Transcriptase buffer composition. Are they similar? Each enzyme will be optimised to work in specific buffer conditions and it's possible that the DNase buffer will be inhibiting the Reverse transcription reaction.
Similar to the poster above, I would recommend an additional cleanup, using a Zymo column to remove the DNase buffer, and any residual DNAse. The columns have a good RNA recovery rate, and I think you can ask for free samples ;-).
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