Hi GUYS,
I am new to this forum ...I am currently using a Qiagen's hotstar Taq Mastermix for my PCR reaction. My question is what is the miminum amount of template DNA beyond which there will be Undetectable amplification product ?
Any feedback would be great !
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need to know minimum amount of template DNA needed for pcr amplification
Started by rwadhwan, Nov 28 2010 06:42 PM
2 replies to this topic
#2
dtae
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Posted 01 December 2010 - 09:21 PM
not sure--but here are a few hints to start thinking about it:
-a new technique called digital PCR tries to utilize the point which the dilution of template is so low that some number of wells won't amplify. if you look up the literature on that technique i suspect you can calculate the theoretical point at which their is likely to be less than 1 copy your template in the reaction
-if you are trying to get by on really low amounts of template, you might try increasing your number of cycles, or even doing a nested PCR
-you could just test this empirically by creating a standard curve
-a new technique called digital PCR tries to utilize the point which the dilution of template is so low that some number of wells won't amplify. if you look up the literature on that technique i suspect you can calculate the theoretical point at which their is likely to be less than 1 copy your template in the reaction
-if you are trying to get by on really low amounts of template, you might try increasing your number of cycles, or even doing a nested PCR
-you could just test this empirically by creating a standard curve
#3
hobglobin
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Posted 02 December 2010 - 04:43 AM
There's no finally answer, though actually 1 template DNA could be enough. But it depends on many factors (most important are IMO primer quality and DNA type/quality, and reaction conditions such as annealing time and cycle number). I learnt that about 100-300 ng of genomic DNA is sufficient for a "normal" PCR (plasmid DNA even less in pg range).
One must presume that long and short arguments contribute to the same end. - Epicurus
...except casandra's that belong to the funniest, most interesting and imaginative (or over-imaginative?) ones, I suppose.
