Hi, I need some advice on long range PCR. I am making a knock-in mouse model and need to genotype mouse ES cells to verify correct recombination of the long arm. The short arm was done by PCR. The Southern strategy for the long arm failed because the designed restriction site is methylated and the enzyme doesn't cut (poor planning by the people who designed the vector). I am looking for a 12.2kb fragment and have tried a PCR approach. I tried the Roche Long Template PCR kit with 9 different combinations of 300nM primers (each pair has nearly same Tm and GC content each 22-25 bp in length), a gradient of annealing temps (60-65, based on Tm of primers) and all 3 supplied buffers. I've used 400-500ng of WT mouse ESC genomic DNA as template. The only products I get are non-specific. Please help!! Roche technical support keeps telling me to decrease the Mg++ concentration and primer concentrations, or re-design primers but I've tried and it doesn't work. Thanks!!
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