PCR with 75 mer forward primer - (Aug/17/2006 )

Hi!
I am new at this (bioforum and molecular biology), so...bear with me!
Here it is:
I am running PCR with un unusual set of primers:forward of about 75bp(containing the melittin sequence) and reverse about 20bp.Have 2 paires of primers:one for TOPO cloning,without restriction enzyme sites,with Tm of 68 and 51, and one with restriction enzymes sites,Tm of 68 and 60 respectivly.
The expected product :about 950bp.
Enzyme:KOD+
PCR conditions:
94°C/2min
35 cycles :94°C/15 sec
55°C/30 sec
68°C/1 min

Tried different concentrations of template(plasmid DNA),different concentrations of MgSO4,adding glycerol,hotstart and when I do obtain something its a band or several of at least 2000 bp!
I know that also the annealing tempearture is critical,but I have been told that giving the lenghts of primers maybe its not so important!However,I did run PCR at 60°C,annealing temp, also.(had results with only the second pair).
Do i need to redesign the reverse primers?make them longer,to increase Tm?
But something thats really bothering me:I run PCR with just one primer in the mixture and with the reverse one got a band of 2400bp;with the forward one:nothing!
WHY?

Thank you for your pacience!

How many cycles r u doing ur PCR for ?How much of template do u use?

Did u check if ur primer might anneal at a similar sequence in the plasmid.

Try 65C for annealing.

I'd consider extending your reverse primers, because 17 or 8 degrees difference is waaaaaay too much! Before you do, however, is the Tm of the forward primer based around the template-specific sequence? If you have included some of the other add-ons in your calculation, re-do it with just the sequence-specific part. I always aim for no more than 5 degrees difference between Tms.

Actually when you run a Megaprimer PCR you have one primer way longer than 75 bp. So I do not think this is a problem. Doing megaprimer mutagenesis I found very useful to add a proofreading taq 1 to 20 with the normal one. Let me know if it is working also for you!

Micky

hi
i've succesfully done pcr at 75° with anneal at 70°.
Do 10cycles with your own tm
and then increase temps for the rest, as the calculated tm of 61 is for the first cycles only, as in the rest of the reaction the whole primer anneal.
You may also try DMSO.
M boss did fine PCR yesterday with 12%DMSO and taq polymerase...