So, I synthesised my first strand of cDNA, and used the above kit to clean it up. However I am now concerned that the kit is for dsDNA only, and that my ssDNA may have passed through the column and been discarded!
Going into cDNA synthesis I had a low concentration of RNA (~10 ng/uL), after synthesis the concentration was up to 400 ng/uL! Then after the clean up this came back down to 9 ng/uL.
So I'm wondering if the high reading was due to left over hexamers (which have now been removed), or whether I've unknowingly discarded my sscDNA when using the kit!
Help is much appreciated!
Hello Cate,
in principle silica + chaotropic salts can be used to purify ssDNA, however, to the best of my knowledge, in standard column-based purification systems salt concentration is optimized for dsDNA recovery. This means that only decently long sscDNA (hundreds of bases?) will be purified, and with low efficiency.
May I ask you why you need to purify sscDNA after RT? what you want to do with it?
Regarding concentration measurements, all types of nucleic acid absorb at 260nm (no real distinction between RNA vs DNA, monomers vs polymers, etc), so your increased concentration after "synthesis" is probably just the NTPs you added to the reverse transcription reaction, which are subsequently washed away during the column purification procedure.
However, I've never heard of an application where cDNA needs to be "cleaned up" for future applications. If you're doing something standard, like qPCR, it is not necessary.
Hi both, thanks for your replies!
We we're under the impression that the kit was for double stranded cDNA and got our wires crossed, luckily I have a lot of left over RNA so all has not been lost
Cate
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