pET32a vector (3 or 1 µg) 28 µL
NEB Buffer 4 5 µL
BSA (100 µg/ml) 0.5 µL
EcoRI-HF (40 units) 2.0 µL
XbaI (40 units) 2.0 µL
H2O 12.5 µL
TOTAL 50 µL
Attached is the gel image. Lanes 2 and 3 are EcoRI only for a linear control. Lanes 4 and 5 are 3 ug vector double digest, and lanes 6 and 7 are 1 ug vector double digest. All digests were overnight. You can see that the correct size fragment is present around 600 bp (200 to 12000 bp ladder) but it is very incomplete
Does anybody have an idea as to why I have incomplete digestion? I think it might be the plasmid prep. The uncut plasmid hardly migrates on a gel as shown in the Picture 1 figure in lane 2. The plasmid could be nicked, and that is why it wont migrate but it still should be digested.
It seems as if no matter how much enzyme I add or how long I digest, I do not get any significant digestion.
Any advise? Tomorrow I will be doing a new plasmid prep.
Edited by HOYAJM, 26 January 2012 - 11:16 AM.