Hello,
I have some troubles with the synthesis of a DNA probe for FISH in paraffin tissues, we decide to synthesize it by PCR with labelled dUTP, but we haven't had any results, I tried chaging primer concentration and modifying a little bit the MT temperature, but nothing works, someone know what might be happening? Could be the type of polymerase or Mg?
Please help!
Thank you
I would run a gradient pcr of annealing temperatures from Tm +4 to Tm -6 with ordinary ntps to get a working pcr then try it with the dUTP added once you know the conditions to use Also are you just using trace amounts of dUTP or is it replacing the TTP in the reaction and what is the label on the dUTP
I replace TTP in a 25% of its concentration. I followed the guidelines of some manuals that are online. Like that in the image.
Yes, I was thinking something similar, I'm not sure, I will try with a different one.
Thank you.
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