Hello. I´m working with a 64bp segment form exon 7 of AB0 blood group gene.
I need to digest the amplification products with enzimes MnlI and NlaIII (Fermentas) to determine genotypes. The problem is that I've tried several digest protocols (the ones suggested by the fabricants, and the ones in Hummel et al 2002 from which we got the techinque, among other variations) but the digestion didn´t take place in any case.
Maybe the problem is that the segment is too short for the enzyme to work?
Has anyone experienced any similar problem?
I´ll apreciate suggestions, comments, etc. Thank you!