re-pcr - (Sep/22/2005 )

hi
i wonder the what are the conditions needed for re-pcr. my pcr product is 1.2kb. im trying to re-pcr(using the pcr product as the template) but never succeeded. i get a smear, what parameters should i change? should i decrease the annealing temp?
Thanks in advance
cheer
chandima

If possible, optimise your second PCR on the first template (annealing temp, Mg,...). If this one works, make sure you don't add too much of your first PCR. Run a gel of the first one, and if it's intense, just use 1/10 of a µl (first make a 1/10 dilution and then add 1 µl).

Becuase it is re-pcr, I'm almost sure that you should reduce the template concentration.
In many times, smears in pcr mean "there are too many ingredients". In most cases, the template is the answer.

hi
thanks for the comments but i tried 10-1 and 10-2 dilusions; 2ul of each dilusion. the first pcr is very intens around 100ngul-1. ill give it a try again
cheer
chandima

If your first pcr is very intense, why do you need a second PCR? Have you tried optimising your second PCR on itself?

Re-PCR is a bad idea/ If you can not see a signal in 35-40 cycles then your reaction is not working well or you must be below 10 copies for detection. In that case it is always better to use a nested-pcr reaction. Be careful though, at that sensitivity your results can reak of contamination. Make sure to do a background control.

hi
thanks, i want the re-pcr 'cos my template dna is messy. I m working with a pathogen in sugarcane. I extract total dna from the plant and and use specific primers to amplify the pathogen. after awhile the dna wont amplify. so if repcr works that is easy for me, im afraid i dont get what tap14 mean, could u pls explain what u ment by " In that case it is always better to use a nested-pcr reaction. Be careful though, at that sensitivity your results can reak of contamination. Make sure to do a background control."
thanks
cheer
chandima

It sounds like you are basically searching for a needle in a haystack. Your pathogen is most likely way below one copy/cell. I see what you are trying to do with your "re-pcr", but it is more commonly accepted in use a nested PCR reaction. This assay uses a second set of primers that recognize sequences within the initial amplicon. The reason to do this is that if you have non-specific amplication in your first reaction in the slightest degree, it will be amplified in your future pcr cycles and may outcompete the traget you are really searching for. I STRONGLY suggest you use this approach or you may end up with a bunch of junk like you are obviously getting.
As far as the contamination goes, anytime you are pushing PCR to the limits, i.e. looking for 1-10 copies, you run into the possibility of contamination. Basically, with conventional PCR, if you "accidentially" introduce a couple of copies into your reaction, you may never see a product on the gel due to sensitivity of the assay. However, with this protocol, any slight contaminant will be amplified.

hi
thanks, i get it now, it seems i might need to plan it all over.
cheer
chandima