I doube digested pET29C with Ndel and Kpnl-HF from NEB. However, after gel purificartion, runing the agarose gel gave me just a single band.
I expected to see three bands represnting the isolate peice of vector around 54 pb and 5373bp which is uncut vector and 5318bp band representing the cut one. But only a single band around 5373 or 5318!!!
Can it be a true result? I mean is it possibly include uncut and cut vectors? Or my sigestion work propely and all vectors are cut?
Tnx in advance,
I'm agree with Mangal singh. It could be both single and double digested linearised vector. The difference between single digested and double digested vector is only 54bp which cannot differentiate with 0.8% agarose gel electrophoresis. Observation of 54 bp fragment along with 5000bp fragment is very difficult, Even u want to observe 200 bp vector piece it is very difficult. You should have to have a high concentrated digested products or combination of many digested products should be load to a single well. A very first 10 min time if you observe the gel you might see the low molecular weight band near to the 5000 bp band. Anyhow there is a possibility to retain single digested vector along with the double digested vector.
