How to do PCR fragment in 2 steps - (Feb/27/2009 )

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?

ups on Feb 27 2009, 12:01 PM said:

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

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

ups on Mar 2 2009, 03:20 AM said:

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.