if i am going to optimize my pcr conditions, where will i start? the annelaign temperature or magnesium chloride concentration or Taq conconetration? thank you.

I would play around with magnesium concentrations first, you know what the calculated Tm of your primer is, set the PCR cuclying to 2 degrees below this and it should work, if not, I will then perform a titration of MgCl2 from 0mM to 3mM and see which concentration works best.

if you have a gradient PCR, you can then perform both MgCL optimisation AND Tm optimisation at the same time!

I would not adjust or play around with Taq concenctrations for the meantime....it wouldn't do much.

Nick

you can also add 2%DMSO to your mix. That will not affect taq activity but reduces the unspecifics.
fred

thanks again. i have been trying to detect three genes in one reaction, ( ranges from 219-450 bp) and some samples amplify and some are not due to the varying amount and purity of my dna. should i stick with the optimized condition i made with the dna sample that amplifies well? or should i continue optmizing until i can get the perfect condition that can amplify all smaples regardless of dna quality?

what are your comments about ammonium sulfate buffers?

I think that if your DNA is of bad quality, you never will get much good PCR results.

it is a big challenge to multiplex PCR with three different genes. You need to optimise each primer set individually and find a common condition for all three, it is another story when you actually do the multiplex, you may get suboptimal amplification of one more more primer pairs, although it sounds very convienent to multiplex, in the long run you may find it would be easier to single-plex your PCR to ensure you get amplification of each gene of interest!

Good luck!

Nick

yes, thank you very much for your input on just doing it on gene at a time. I have consumed so much already with multiplexing and i get not that intense bands. I guess i have to detect the genes individually.... thanks again for your input. regards

Optimization of PCR is tedious job. First of all, if you run multiplex PCR you need to check primers' melting temperature and play with PCR conditions. Than take to account their concentrations as well as their ratio. Taq DNA is common reagent, but it lucks proofreading activity. Recombinant Taq DNA Polymerase is another option. MgCl2 is reaction component with which you can start, but excess of later could "backfire". Run each one separate and than combine them both together. Are there any inhibitors in your sample? Inhibitors present in sample can influence on your reaction outcome.

Normally multiplex PCR is a nasty task if you ask me, especially when you use the normal polymerases. BUT, I recently tried the Qiagen Multiplex PCR kit and suddenly I could plex alot of primer pairs. So if you have any trouble then try this kit, you could ask for at testsample first to see if it solves the problems. I am NOT from Qiagen