I understand a normal really simple PCR reaction. But now i have a small problem.
My gene:5' (Rest of genome)TTATCCT........TACTCAT(Rest of genome) 3'
In my vector there are the following restriction sites:
HindIII5'-A|AGCT T-3' 3'-T TCGA|A-5'
XbaI5'-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
Even I would suggest you to attach a restriction enzyme to your designed primers and then amplify the plasmid and ligate it. So, the answer to your third question is 'Yes'. Also, make sure that the enzyme should be added at the 5' end of the primers.
The primers should be between 25 and 45 bases in length, with a melting temperature of ≥78°C. The desired mutation should be in the middle of the primer with ~10–15 bases of correct sequence flanking both the sides. And both the mutagenic primers should anneal to the same sequence on opposite strands of the plasmid.
Here is a freeware to design specific mutagenic primers: