I isolated mRNA from HEK293T cells using oligo dT cellulose, followed by RNA Cleanup and Concentrate kit that includes a DNAse digest. The final mRNA yield was about 6.7ug, with 260/280 and 260/230 values indicating clean mRNA. I also ran a sample of it on a denaturing agarose gel (image attached).
I used ~3.3ug of mRNA in the Maxima H Minus Double-Stranded cDNA Synthesis Kit, including the RNAse digest at the end to remove potential RNA contamination. ds cDNA was purified using a PCR Purification kit. Final cDNA conc. was 100ng/uL, with a total yield of 3ug. 260/280 and 260/230 values were good, but I am puzzled by how my cDNA runs on an agarose gel (1% TBE - gel image attached).
Why is there a predominant band around 600bp?
UPDATE: I re-ran the sample using a different loading dye that does not contain SDS, and cDNA runs as a smear.
Do you add the dye/EB in the gel? if so, you might not see lots of DNA running slower (most of the dye/EB bind to the DNA running fast). you could try to stain the gel after you run the gel.
I think it is unlikely you can get useful information about cDNA quality from an agarose gel and the best way to measure your cDNA quality is to do PCR.
Maybe it depends on the speed of the transcriptase and it does not create full transcripts? If that is the case, you might try longer incubation time for RT
Junjie Shao thank you for your suggestion! I actually did not add the dye in the gel, I stained after running. I re-ran the sample in a loading dye that does not contain SDS, and now cDNA runs as a smear. So the intense band around 600bp must have been a loading dye artifact.
Jafar Nouri Nojadeh I see, thank you, will do that!
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