I wanted to clone my PCR insert into pET-28 vector (which was isolated from BL21 DE3 with kit) between BamHI and XhoI sites. For 4 hours I digested vector and insert in two different tubes (25 ul of total volume, 5 ul Tango buffer (x2), 2 ug of insert, 2 ug of vector, 0,5 ul BamHI (5U), 0,5 ul XhoI (5U). Then I runned 3 samples on 1,5% agarose gel: 1-double digested PCR insert, 2-double digested vector, 3-undigested vector (0,5 ug). I see one band of 1 (correct), two bands of 3 (correct) and there were no bands at all of double digested vector, totally nothing. Why is that? Does anyone know what could happen? I found similar topic but I couldn't find the answer. Thanks for help!
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No bands at all after double digestion
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