DNA - (Oct/20/2001 )
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 ?
maybe try to reduce the annealing temp with the system 1 component of the expand kit until you get product. (hopefully it will be your target sequence) Or just design new primers. PCR is like Voodoo or magic sometimes. Something that seems like it is supposed to work may not work, but something that you would think would never work may give you what you want.
How much time do you live for the replication of this long PCR?