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Touch Down PCR


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#1 Microbes in action

Microbes in action

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Posted 19 February 2011 - 04:55 AM

I have a good PCR products with a good sensitivity with this T.D.PCR cycle.....

95c - 6mins

95c-50s
67c-1min - 3 cycles
68c-2mins

95c-50s
67c-1min - 8cycles (reducing cycle, 1c decreament in each cycle, so it reaches 60c)
68c-2mins

95c-50s
67c-1min - 29 cycles (normal)
68c-2mins

72c-5mins

My question here is, I'm running the PCR again with a high annealing temperature of 67c, usually in a T.D PCR it should be the lowest temp that was achieved in the decreamenting cycle, which would be 60c in my case. On the contrary, if I do the same samples with the final 29 cycles with annealing temperature at 60c, I have less sensitivity. My samples are dilutions of PURE DNA. Say for example, I get product formed up to 10^6 in the previously modified T.D. PCR cycle, but in the regular T.D.PCR cycle, I was able to get only 10^4.


Would some one explain this T.D.PCR phenomena.....




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