my insert is there by PCR check but no band after digestion... - vector not digested because of contamination? (Jan/11/2006 )
hello all again,
i have done PCR to amplify RE sites and inserted my PCR product into the vector, checked by PCR the E coli colony and faint band of insert was there....so I multiply E coli again and do double digest to check the insert .....and it seems as if whole vector is not digested, there is no insert and control and double digest samples have similar bands on the gel....(not digested?) ...could my sample not be digested because of contamination? what should I do? where could be a problem?? any help will be appriciated
I had that problem.
To check the insert is there, use restriction enzymes that flank your insert. ie ones further out than the ones you used.
Sometimes, durng the ligation process mutations happen when the insert is inserted. the result is the insert is there, but can't be detected using the same REs used to cut the insert and plasmid.
I didn't PCR the plasmid to check the insert first though, i just double digested it... and sequenced it.
Have you PCD'd any of the controls? just thinking the problem might also be linked to the PCR process.
Hope this helps.
thanx a lot.....unfortunalty i dont have any flanking sequence outside the insert so....im PCRing it again the one that i have extracted and if it doesnt have the piece than i guess ligation was weak or something.....strangest thing is that yesterday i ran the digestion again using 2 control vectors that i have used two weeks ago and put at -20.....one of the control is ok but the second one was not digested same problem as my recombinant one... ...very very strange...what could happen to my control vector now?!?! im just so confused....
Could there be a problem with your restriction enzymes? perhaps they're dying?