After digesting vector with AsiSI and NotI, I gel purified and quantified. The insert was PCR amplified, gel purified and cut with same enzymes. After cutting insert, I did not gel purify but heat inactivated at 80C for 20min (as recommended by NEB company). I didn't get any single colony after ligation (1:3). I was just wondering that is it possible that enzymes are not properly heat inactivated and still actively cutting the ligated product? Any tips for successful cloning will be appreciated. Thank you ~

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