mirna overexpression/ library versus single construct/ unspecific pcr - unspecific band when pcr on gDNA from library but not on gDNA fron sin (Jan/05/2011 )
I am using lentiviral mirna overexpression library. In order to detect mirna inserted into genomic DNA, I use the primers that were included in the manual coming with the library.
If I do a pcr on a genomic DNA from cells transduced with a single mirna construct, I obtain nice clear sharp band.
If I do a pcr on a genomic DNA from cells transduced with library, of course, I expect to obtain many products and many different sizes since inserted mirna constructs differ in length ( but the product still should be within range 500-600bp). Unfortunately, I see on gel additional band of size around 300bp. I do not understand why I see it only with library but not a single construct, overexpression plasmid is the same...and how could I get rid of this unspecific band from my pcr. I tried temperature gradient and the band gets weaker but still there...
I will be very grateful for any advices!
Are you sure the unexpected ~300 bp band is non-specific, not from some shorten miRNA constructs? How about sequencing the band to find out?
Hi! I contacted the manufacturer and they say that actually there can be premirna constructs of 300bp, so now I will definitely cut it and sequence. All my confusion was coming from the fact that the manual says that premirna constructs are between 500 and 600bp so I did not expect that there should be anything smaller than that. THanks a lot, now I have 2 independent advices: lets do a sequencing!