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How to avoid nonspecific amplification (band) in PCR

PCR non-specific

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5 replies to this topic

#1 Inbox

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Excellent

Posted 26 July 2013 - 06:59 AM

Hi,
How to avoid nonspecific amplification in PCR from PCR?

thanks.

Edited by prabhubct, 26 July 2013 - 07:00 AM.


#2 bandeyabo

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Posted 26 July 2013 - 05:48 PM

check the magnesium concentration you used as well as annealing temperature. Usually high mg+ results in non specific amplification

Edited by bandeyabo, 26 July 2013 - 05:49 PM.


#3 christy

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Posted 28 July 2013 - 05:14 PM

Gradient PCR to set specific Tm for the target gene.



#4 mdfenko

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Excellent

Posted 29 July 2013 - 04:15 AM

very specific (and long?) primers


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#5 Ameya P

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Posted 15 August 2013 - 10:06 PM

higher annealing temperatures help too


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#6 hobglobin

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Posted 16 August 2013 - 08:09 AM

Several additives such as:

dimethylsulfoxide (DMSO)

glycerol

formamide

betaine

tetramethylammonium chloride (TMAC)

polyethylene glycol (PEG)

can help but not necessarily.

Some are also seen as additives that facilitate the reaction, increasing yield or work against inhibitors, the functions seem to differ between authors and are somewhat difficult to define precisely. So it's trial and error, as with the recommended concentrations.


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