Hi,
I had set up PCR with 50ng of FFPE and i was able see band at expected size. when i tried to repeat the same PCR with 50ng each in 4 vials i:e to bulk up the product. i did not get any band though my positive and negative worked. Can somebody suggest the optimum concentration what i have to use .
Hello Sravanthi
50 ng is enough quantity , but i think that you have to care about two inhibiting reasons :
first : the presence of the inhibitor , for this check the 260/280 and 260/230 ratios and can be over come by re purify the DNA .
second : the DNA integrity , check the isolated DNA integrity by agarose gel electrophoresis , if you will see a large quantity of small molecular weight fragments ( smearing ) this is very bad for PCR , in this case you need to use another DNA isolation protocol or purify your DNA by gel .
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