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I could really do with some help! I have general primers that ought to bind to the cytochrome b mitochondrial gene in bos taurus (cattle). now, the forward sequences have worked alright 2 out of 3 times, the reverse hasn’t workerd at all except in one single sample during the very first run!? i know my initial PCR is ok so it has to be the sequencing cleanup or the sequencing pcr reaction. i get either no run at all, just 4 or so unidentified nucleotides or i get huge dye blobs and G due peaks that stretch for many bases. could it be the cleanup step when my forward reactions work well? could it be the primer? some outside thoughts would be appreciated! feel free to E-mail me at s24061621@tuks.co.za
kind regards
anandi
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