Hello folks,
I am working on T7E1 assay and I found non specific band in my samples while wild-type DNA has no cut. I amplify my fragment using Q5 Hot Start 2x master-mix. I use 1 ul T7E1 enzyme in a 20 ul reaction containing 200 ng purified PCR product. Incubate the reaction volume for 15 min at 37 Degree Celsius.
are both fragments "single specific bands" on agarose gel before adding the T7E1 enzyme ?
Hi. Moahmed
I hope you are doing well, to a void the off target of T7E1,
1- your PCR products preferred to be less than 1 kb avergae 800 bp is okay
2- usually after annealing using NEB buffer 2 the PCR product shifts on gel about 150-200 bp (if your orginal PCR is 800bp after annealing and T7E1 digestion the PCR will be around 1Kb) so make sure that your two fragments after digestion will also shift the same ratio.
3- if the size of the band is visible and can be purified you can sequence it. then you can judge wither its specific or not
best of luck
Hi Muhammad Salman Mubarik
I think you must recheck your primers specificity and design, also you must not forget the possibility of contam during isolation..
Sometimes your own little modifications give best results than standard protocols.
good luck
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