Electrophoresis problem - (Jul/07/2007 )
My vector and insertion were double digested with the same enzymes and gel purified.
After ligation and transformation I have got a lot of colonies.
I picked ten of them and extracted the plasmids.
However, the electrophoresis result is amazing to me:
the plasmids that I extracted run even much faster then the control vector(see attachment, lane1 and lane7 are controls).
Anyone if you have encountered such a problem?
Thank you!
normal
the ligation - transformation product are more big than control so it miograte slowly
good luck
could you please give more explanation...e.g. please explain if the elecrophoresed plasmids (extracted and/or control) are digested with restriction enzymes or not? if your extracted plasmid contain an insert .. so what is the molecular weight?
where is your marker in the gel?? i mark the gel in the opposite way that you used here
the elecrophoresed plasmids were not digested.
Plamid: 4.5 kb
insertion: 9.6 kb
I did not utlize any marker.
Is`t the Vector plasmid as a control enough?
there seems to be a contamination. a smaller plasmid might contaminate your plasmid prep or insert prep or ligation rxn or etc. 
meaninggg your positive clones are false positives 
Plamid: 4.5 kb
insertion: 9.6 kb
I did not utlize any marker.
Is`t the Vector plasmid as a control enough?
To confirm if there is insert, digest it. Undigested plasmid can run differently. Digest it and then comapre sizes with a DNA ladder which would cover the sizes you were expecting.
What would be the size of the original vector?
My original vector is 4.5 kb.
your discussions will be great helpful for me.
I am going to digest my plasmids.
Thank you all!
please i want to know the result of your restriction treatment. actuually if there is no contamination correct vector and insert size will be seen after restriction. as the ligated form show great variations in the migration through the gel
Yes.
I have checked the size of cut and uncut Vector by agarose gel electrophoresis.
The size of the cut vector is normal (4.5 kb), and to my surprise, the uncut vector runs much slower than the cut vector.
In fact, Lane 6 is the correct construct but still faster than the uncut vector (attachment).
And lane 2-5 is recircularization. It is intriguing that the electrophoretic mobility of the recircularization seems normal. But the recircularization and the control vector should be the same thing. The only difference between them is that one is Mini-prepared and the other is midi-prepared. Would these make it have different conformation?
Really puzzled.