What are the criteria for "good" primers in a PCR reaction?
Describe how you would use site-directed mutagenesis to change a BamHI restriction site into an EcoRI site.
These are the criteria for "good" primers in a PCR reaction.
i. Use a GC content of around 50%, ie don't have too many of one kind of base or your primer may bind repetitive DNA. Don't use runs of single nucleotides.
ii. Use a G or C at the 3' end to make sure base pairing is strong at the critical 3' end
iii. Use a primer length of 16nt or more, though this varies a lot with template. I usually use about 20nt. DNA has 10bp per turn so imagine the primer ...
Criteria for designing PCR primers are listed. A brief description of the site directed mutagenesis needed to change a BamHI restriction site into an EcoRI site is shown.