Generally we perform PCR with 50-100ng of DNA, High conc of DNA may leads to more non specificity and reduced efficiency.
all depends on copy number of your inteded target primer-binding sites versus non-intended, the game of affinity (mismaches) ...the answer is complex but there is no simple answer exept to try
Hi, I normally run my PCRs with 20ng. This amount is sufficient. Cheers, Nadine
It really depends on the template and annealing temp. 200ng of a low complexity template like a plasmid will likely lead to a tremendous amount of non-specific binding unless you have the annealing temp set well above your primer Tm-- enough where your gel would probably look like a smear. With normal sized cloning plasmids, 50 pg is plenty. (Yes- picograms). With increasing complexity, you can tolerate greater amounts of template. With bacterial gDNA I use 1-20ng, with fungal DNA 10-100ng with large genomes like maize, up to 250ng isn't a problem.
The key is to think of it in terms of the number of copies of the target sequence rather than the concentration of DNA. Starting with 1k-10k copies of your template is plenty to give good yields give good yields.
200 ng in a 100 uL qPCReaction? 50 uL, 25 uL, 20 uL or 10 uL reaction?
Here you see you would have 2 ng/uL (which is OK), 4 ng/uL, 8 ng/uL, 10 ng/uL and 20 ng/uL, respectively.
I would suggest doing a serial 1:2 dilution study (20 or 30 points) of your DNA and see how the reactions behave. If you have too much target in the beginning reactions, you will see template inhibition of the reaction(s) (Cq values higher at first, then they will get lower, and then start responding to dilution correctly).
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