How do I know if my cDNA are of good quality? - (Jun/13/2007 )
How do I know if my cDNA are of good quality?
Run them on gel. You should see 2 clear bands, one a supercoiled one and another relaxed one. If there is any sign of smear, probably there is contamination in your samples.
scolix: You can see cDNA on gel?? I've never managed to get this (there are RNA-DNA duplexes usually, though).
We perform a control actin pcr to check the quality (but probably won't tell you that much).
We perform a control actin pcr to check the quality (but probably won't tell you that much).
what bout the nicked DNA?
We perform a control actin pcr to check the quality (but probably won't tell you that much).
what bout the nicked DNA?
I guess the nicked DNA would also run similar to the relaxed state DNA.
But I have single-stranded cDNA, can I somehow visualize it on a gel?
And should I not get a smear? (I used oligodT making all mRNA to cDNA).
Hey there,
yes, you will probably get a smear. As far as I know, the only way to assess cDNA quality is to run a PCR using a housekeeping gene or a standard gene, and compare the results each time. If you're worried about the efficiency of the RT reaction, you will also be doing RT controls no doubt, and you can assess them using PCR.
If you do the RT reaction using high quality RNA that you have checked for impurities and quality (using nanodrop/bio-analyser/gels/spec etc), and you know that your RT reaction has worked (based on the RT control), then you can be fairly sure that your cDNA is fine.
actually, there are too many control steps you need. if you want to check every steps i think you will go mad.
i agree with what smurray mentioned.
you can check your RNA quality. if you done well, the rt reaction is reliable to get good cDNA.