I understand a normal really simple PCR reaction. But now i have a small problem.
5' (Rest of genome)TTATCCT........TACTCAT(Rest of genome) 3'
In my vector there are the following restriction sites:
5'-A|AGCT T-3' 3'-T TCGA|A-5'
5'-T|CTAG A-3' 3'-A GATC|T-5'
The promoter is before the HindIII restriction site.
Now i have to create primers to add sticky ends to my GOI to integrate the reverse complement in the right direction.
1. How do you do this? With programs?
2. It doesn't matter if there are hair pins or anything else. My intention is to understand it.
3. Are these primers correct?
5‘ TCTAGA TTATCCT 3‘ 5‘AAGCTT ATGAGTA 3‘1. part = restriction site for sticky ends
Edited by zzz2, 18 October 2012 - 01:29 PM.