primer designing for point mutation?

Question

tryin to design a primer,which shud make point mutation, but the melting temperature is coming at 65 degrees, but it has to be more than 78 degrees....how is that posible without further increasing the G C content?....

Answer 1

There are several different ways to do it.
Personally I use a very simple protocol

5'-10 bases-mutation-30 bases-3'
3'-30 bases-mutation-10 bases-5'

It could be 28-32 bases after the mutation, as long as it ends in c or g (but not more than 3 g or c in a row)
There are other protocols with smaller primers, but this one has never failed me (by the way I use Tanealing =62-64 depending on the template. If you have a difficult template you might want to consider other protocols.

Oh, and after the PCR use DpnI (incubate 2h at 37) to destroy the template DNA.

Answer 2

Hmm...not sure I understand. When I was designing a mutation, I just bit the bullet and bought a mutagenesis kit. They are expensive, and you might be able to put together everything by yourself, but it IS a lot easier, and since the technology is now about 20 years old, it is a lot more reliable. They will also provide for the destruction of the WT strand, so that you don't have to go through as much trouble trying to find a clone that is actually mutant. Also, I don't seem to remember there being any problem with the melting temp of the primer chosen.
Any of the major enzyme sellers should have their own kit (even Sigma has one, if I remember correctly). I would suggest going to their websites and looking for a protocol that you can download, and it can tell you whether they do have any special requirements for a primer.

Answer 3

are you sure the melting temperature of your primer has to be 78?

what are you doing taht requires it to be that high? mine are usually around 60, as low as 45 and the pcr works fine.