Hello! I have been trying to produce IVTs from gBlocks, starting amount is ca. 100/120 ng of DNA.
When the reaction is done, I do a DNAse step, purification on column and then go to Nanodrop. When I measured the samples, I had a concentration of 1000-2000 ng/ul and a A260/A280 of more than 2.20.
I found this very weird, so I repeated the DNAse step on the IVT sample and purification again, but the situation didn´t change. I then did a third DNAse step, with a different DNAse, and again purification but even so I always have similar results.
My question is, can it really be that I have this much RNA? How can I check whether in my sample there´s RNA, DNA or a mix of both?
Thank you!
Depending on the volume in which you eluted your RNA the concentration is not out of the realm of possible.
However, you should remember that nanodrop will also detect small fragments and possibly even nucleotides carried over from the template. You can measure your RNA using Qubit which is specific and will only detect the RNA.
You should also run a little bit on a gel to verify you get a clean band at the right size.
Hi there,
Instead of talking about concentration, go for the total amount of RNA. If after 3 rounds of DNAse treatment and column purification you get a signal, you can be pretty sure it is RNA.
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