annealing temp for degenerated primer - PCR problem (Jan/22/2002 )
Hi all, it's me again. This time I bring another problem. Last time, I performed touchdown PCR, annealing temp started from 48 C and decrease 0.5 C in each cycle, 20 cycles. And I found nothing but primer dimer. I think I will lower annealing temp from 48 C to 40 C. Will it work? Please give me some suggestion. An dI think I will try to vary MgCl2 conc from 1.5 to 4.0 mM. What do you think?
PCR primers should be free of significant complementarily at their 3'-termini as this favours formation of primer-dimer which reduce product yield. Formation of primer-dimer artifacts may also cause more serious problems, such as non-specific DNA synthesis (asymmetric PCR). (Help from the internet: http://www.basic.nwu.edu/biotools/oligocal...ml) Magnesium
Magnesiumchloride is one of the main variables. It affects the annealing temp by stabilising the oligo-template interaction and it also stabilises the replication complex of polymerase with template-primer.
I hope it helps you a little bit.
Are you sure there is enough cycles to see an amplification on gel. 20 cycles is not a lot, depending on the DNA quantity you used.
For the magnesium chloride, I already tried that with difficult PCR and it never worked, it only gived more non-specific products. A good thing to try is 5% DMSO. DMSO is a denaturing agent that helps to eliminate non-specific interaction.
There could be a lot of problems here. Do you have a positive cntrl? What is the melting temp of your primers given the salt concentration? general rule of thumb is to start 5 degrees above temp and cycle to a few degrees below. Could be your dNTPs have gone bad. Agree strongly with cycling 30x as stated before.Aalso touchdown is hard on polymerase, may want to shorten melting time and temp.
Well, I tried searching books and websites and I found one protocol but I couldn't get it coz it was published on science on 1986, such a long time ago. Could you post me a protocol using DMSO as denaturing agent? Thank you.
I think your primer dimer formation should also happen due to the high primer concentration versus low template concentration. I work with ancient DNA - its concentration is somewhere in the pg/ul scale and I use 5pmol/35ul PCR reaction - just to show how low can be the concentration of primers in PCR, try it
Have you tried using a gradient PCR machine? It'll test a heap of different temperatures at the same time.
I'd use more than 20 cycles.
With DMSO, i use that too!!! it works well with my stuff. I add 5% of the final volume of DMSO to the eppi tube when i'm adding everything else in. ie, for a 25 uL final volume, i'd add (where's that calculator...) 1.25 uL of DMSO.
stick it in the machine, swear, kick, curse, and it'll work like a dream.