My Transcription Reaction Failed ... Now What?

Hi Gwen,

First, sorry to bother you on the weekend!

My group performed the PAGE protocol today and we did not get a band. I know that we're supposed to run the lsPCR products on an agarose gel to confirm that we actually had DNA to begin with, but this morning when I was adding the DNase to the transcription reaction, I accidentally added the DNase to the leftover lsPCR reaction instead. So now we have no PCR product.

What should we do now? If need be, we have no problem coming back next week and redoing the experiment.

Also, on the questions at the end of the protocol, number 4 asks "What was the concentration of RNA in uM at the end?" How would I do that? Should I just do a theoretical case and show how to do the calculations?

Again, sorry to bother you on the weekend.

Thank you,

1 comment:

Gwen Stovall said...

(1) For the report, borrow another group's numbers & do the calculations using the RNA concentration that found on the nanodrop. Please be sure to write this up in your report.

(2) Next week, repeat the lsPCR & then the transcription reaction. This should be fairly easy, as you previously determined the number of cycles required for the lsPCR. Have a mentor watch you setup the transcription reaction.

Best wishes,