Help with ligation - (Jan/18/2012 )
I have double digested my pSUPER vector with bglII and Xho I, I run on the gel and the digestion appeared to be working well. but when I do ligation followed by transformation, I do not get any growth. I am sure nothing is wrong with ligation procedure because I did the transformation with an old digested vector and it did work there and I got colonies. It appears that something is wrong with the vector I made, it just doesnt work, I do not know if it is due to damage by UV while I was extracting the vector from gel. By the way I used the restriction enzymes from two different companies can any one help?
I formerly experienced failure with ligation, and it was probably because the insert was too long. I suggest you confirm your ligation step first before transformation. You may do it by cutting with enzyme and check the band size.
try to heat the vector and insert separately at 50 degrees for a couple of minutes, that helps sometimes to arms of vectors free and accessible for efficient ligation.