Please note the new version of primer3 doesnt not give the same primer Tm’s as the earlier version which we know works well. The primer Tms in the new version can be 2-3 degrees higher or lower per primer creating a difference of 5 or so degrees between the forward and reverse (when the standard we use is 0.5C). This will affect how well your PCR product amplifies and what PCR annealing temperature should be used.
Avoid using the new version (Primer3web version 4.0.0) – the top webpage with a google of “primer3” –
Use the old version = primer3 version 0.4.0 (use this – it is known to work well)
It was on this website
but if that webpage is not active (or you havent got it bookmarked) –
the same (good) version is provided here =
or here (again this is the good version, but do check)
Please come and chat if this isnt clear.
Id recommend recording which Primer3 version you use (and the webpage) and also state this in papers.