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Hi fellow researchers!
I have some problems with the separation of two fragments on an arose gel. It would be nice if somebody could share some tips with me.
I want to digest a vector to cut out a small fragment. I have to cut with two different enzymes, the backbone is 7500 bp, whereas the backbone after insert removal is 7000 bp. So I need a good separation on the gel to separate and elute the digested vector. The problem is that I get a weird kind of smear, that I’ve never seen before. That makes it impossible for me to separate the fragments properly.
Does anybody know where this could come from? Here are my digerstion conditions:
1 yl vector (=1303ng)
2 yl buffer Cut smart
0,5 yl NheI
0,5 yl XhoI
16 yl dH2O
I load it together with 6x dye onto a 1% Agarose gel and run it with 100V for 4h. The digestion itself works, but the separation fails.
The very left band is an undigested control, in wich only one enzyme was used. The smear hinders me from separating the bands.