Posted 20 October 2001 - 09:00 PM
I have problems amplifying a 17kb fragment of human genomic DNA of the LDLr gene. I have synthesize 30 bp primers with a Tm of 69 and 68 degrees with oligo calculators which uses a different method of Tm calculation instead of the 4(G+C)+2(A+T). Which methods of oligo Tm calculation is more accurate ? I have been using Roche's Expand Long Template PCR system, I get nothing with system 2 but lots of non-specifics with system 3 even at Tm of 68 degrees. I've tried different MgCl2 titrations but with no improvements. Can anyone help ?