Protocol Online logo
Top : New Forum Archives (2009-): : Molecular Cloning

No bands at all after double digestion - (Jul/24/2012 )

Hi there!

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!


I suppose it is a simple mistake in loading or anywhere. Just repeat it from the beginning and see if the problem is still there. Be careful with the loading and also check if the wells in your gel are alright.