Dear researchers,

I have some problems with my bands on the agarose gel after enzyme digestion. I want to cut my insert (3800bp) out off the vector backbone (5500bp) and perform gel purification. The digestion itself worked very well, but the bands on the gel look some kind of weird and fuzzy. We have already cut out the insert on the gel, thats why only one band is left on the example picture, but the 2 bands of vector and insert look exactly the same. We digested 10ug DNA over night with KpnI-HF (5ul of 20000 Units/ml) (smart cut buffer) in a volume of 50ul as we need a lot of DNA for further experiments. We also performed some days ago a control digest with lower amount (300ng) and those bands look normal and digestion also looks good. We run a 0.7% agarose gel. Does anyone have any suggestion? Is 10ug DNA too much in 50ul reaction volume for digestion?

Thank you in advance, best regards

Heike

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