Amount of DNA template in PCR reaction - (Aug/21/2005 )
im very new in doing PCR so i hope you can help me. how much is the suitable amount of DNA template i should put in 50 microliters of PCR solution?Also, do i have to put exactly same amount of template for every sample?
thank you so much!
Depends. If it is genomic DNA a range of 50-500ng is reasonable. If it is plamid DNA then I would suggest 100pg-1ng range.
If you want to quantitiate the results and compare them to each other, then putting in the same amount of starting template is probably a good idea.
I would suggest you use up to 500 ng with genomic DNA and bout 50 ng with plasmid as a rule of the thumb. But take care to increasethe amount when you use large plasmids, (bigger size, less Sequence of interest per ng).
More important are variables like annealing temperature and elongation time.
I had the best PCR-Results for Cloning with PfxPlatinum polymerase.
Hope I could help.