I produce CRISPR gRNA by using PCR method. I obtained 10ug of RNA from in-vitro transcription. But when I proceed to gel electroporesis analysis I can see any visible band. There was almost blank. Why is it so?
Hi, question is not very clear on a few points.
1. Of course PCR does not produce RNA. So, do you mean you have a PCR fragment containing a T7 or SP6 or T3 promoter at the 5' and you are the using this DNA for in vitro transcription?
2. How do you know you obtained 10 ug of RNA? If you measure nucleic acid concentration with a spectrophotometer, the reading will be the sum of your DNA template (DNAse digested or not) and all non incorporated ribonucleotides, even if in vitro transcription did not work... The way to quantify the RNA produced is with an agarose gel or with the Qubit, where you use the specific dye for ssRNA.
3. If you don't see anything on agarose gel (I guess a 3% is safer, you should have around 150 nt for a sgRNA) I would say the in vitro transcription did not work efficiently. Apart form the fact that T7 and SP6 seem to be better promoters compared to T3 in the general experience, and assuming you introduced the correct promoter sequence, it might simply be how you purified the PCR fragment, or it might be the in vitro transcription kit you happened to use (gone bad, expired, RNAse contaminated, etc).
Good luck.
Hi Pietro,
Thanks for your time.
I will look through all your suggestions and of course will repeat with 3% gel electrophoresis.
Hi,
Are you running a size marker on the gel in parallel? And is that resolving ok?
If you routinely use RNAse in you DNA preparations (or anyone in your lab) residual RNAse in the gel tray can destroy most of your loaded sample as it is running. When I was doing ivt to make mRNA I would only get s smear on the gel where I loaded the marker and see nothing in my sample lane, despite an ok Nanodrop read and electroporation.
Simply properly cleaning the gel tray and using freshly made up buffer with DEPC-treated water fixed the problem for me. Also, it seems that voltage shouldn't be higher than 10V/cm.
Good luck!
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