smear in PCR - No consistancy in amplification (Jun/13/2006 )
Hi frnds
I m trying to amplify a 4 kb gene from rice but if i set 6 reaction at a time with same component & temp, i get amplification in few & in others its smear. i also tryied it with different thermal cycler but there is no consistancy in my result. wat my b the reason plz help me.
do you buy or make a mastermix?
do you vortex your buffers and such before you add them?
are you certain you're not seeing degradation? are your tips nuclease-free and filtered, and is your pipettor 'PCR-clean'? these things could cause sometimes-degradation...if it were your water or dNTP's or something I would suspect more uniform trouble....what about your primers? are they old?
do you vortex your buffers and such before you add them?
are you certain you're not seeing degradation? are your tips nuclease-free and filtered, and is your pipettor 'PCR-clean'? these things could cause sometimes-degradation...if it were your water or dNTP's or something I would suspect more uniform trouble....what about your primers? are they old?
i make mstermix
and do not vertex buffers
their is no degradation bcoz smtime i get amplification by same mixture.
I would not assume that there is no degradation because it works sometimes
these things don't degrade all at once autmatically, it takes time
what about your pipettors and tips?