I have ordered platinum Super Fi II mastermix from thermo scientific for this long range PCR. i am using the primers from Bagnall et al 2005. now i am getting the 14 to 15 kb band in place of 10kb product. moreover i got many different sized light non specific bands too. so if anybody using this mastermix for this assay, or any other mastermix or enzyme and mixture, then please help me to set up this. thank you in advance.
Assuming that the 14kb band is quite strong have you tried a restriction digest to check the fragment size.It may be that either the dna ladder or sample is running oddly.Is the large size generated from a single sample or have you run more than one different samples and always got the larger size amplimer?
I have run a 1kb ladder which provides 5kb to 15kb bands. And i have checked 4 different samples. In all those i found the same results.
When 4 samples all give the wrong size with previously tested primers I am still tempted to say the amplification is right but the band is running at the wrong size.If you have enough product and a suitable enzyme it would be interesting to see if all the expected bands are present and add up t the right size or even run a known amplimer or other size stabdart to check a different size band against your ladder
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