Hello guies!! I am quite new to this work. m optimizing a nested PCR protocol in which i am encountering few nonspecific bands along with the required PCR product. Please help me to know where the problem lies?? Is my Mg++ conc. is high?
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Ameya P
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Posted 29 June 2011 - 10:03 PM
Please provide a picture for your problem. It makes it easier to make suggestions that should work.
Reducing Mg++, increasing annealing temp, increasing/ decreasing extension time and temperature are different suggestions that can be made depending on where your un specific bands are.
For bands lower than your band of interest, increase annealing temp, for those higher, decrease extension time.
Reducing Mg++, increasing annealing temp, increasing/ decreasing extension time and temperature are different suggestions that can be made depending on where your un specific bands are.
For bands lower than your band of interest, increase annealing temp, for those higher, decrease extension time.
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