My negative control is showing bands as in positive control. I repeated the experiment with new reagents and new primers. However the pipette used for adding template was used for gel loading also previously. Although I have been cleaning pipettes and all the instruments with DNA zap and using filter tips . The problem of amplicon contamination is not going away . What is the probable source of the amplicons and how it can be tackled?
Hi,
You may have to do a careful analysis in such a situation..there much be some contamination...sometimes even in the polymerase vial you might be using over a period of time.....use multiple controls to figure it out
Thank u. I will do trouble shooting by using new reagents in a different lab.
For an immediate resolution of the problem; replace each and every reagent including the positive and negative controls.. It may be because your negative control is contaminated with positive one... while handling.. Is seems to be most probable cause..
After that the resolution lies with the handling of pipettes and cross contamination...
....Check the primers which you are using .. for the specificity... they should be longer than 25nt (when designed ) .. and lastly also check the Taq Pol for the contamination...
Hope it will be of some help... Regards
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