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How to optimise touchdown-PCR

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I am currently trying to reduce some nonspecific bands from my PCR and tried a touchdown PCR, which worked quite well.

What I want to know is, how to I improve this specificity? I.e. How can I optimise my existing TD-PCR protocol? Although the PCR works, my negative control template still produces nonspecific bands.

My reaction is:
95oC for 3 mins
10 cycles of
- 95oC for 30s
- 70-60oC for 30s
- 72oC for 60s
20 cycles of
- 95oC for 30s
- 60oC for 30s
- 72oC for 60s
72oC for 10 mins

Thanks!
total 233 hits     Asked by: Anonymouse I want to answer  


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