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How to do PCR fragment in 2 steps


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#1 ups

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Posted 27 February 2009 - 03:01 AM

How can I do that? I cannot do a single step as there is a too long primer ( with a long hanging tail) and I need to have a blunt end:(

Please any suggestions?

#2 scolix

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Posted 28 February 2009 - 08:15 AM

How can I do that? I cannot do a single step as there is a too long primer ( with a long hanging tail) and I need to have a blunt end:(

Please any suggestions?



What is the size of your primer and what is the size of the DNA to be ampified?

#3 ups

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Posted 02 March 2009 - 03:20 AM

The primer is about 70 nucleotides and the fragment that I want to amplify is 3000...

#4 WHR

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Posted 02 March 2009 - 07:15 AM

The primer is about 70 nucleotides and the fragment that I want to amplify is 3000...



Hi,
Assume that 20 bases of the 70 is complementary to the template, you may use lower annealing temperature for the first 5 cycles to allow primer binding, and higher temperature for the rest of cycles.

Edited by WHR, 02 March 2009 - 07:15 AM.





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