BAC cloning problems - (Feb/18/2005 )
I am trying to clone viral genomic DNA into BAC vector (pCCBACI by Epicentre).
I had no success so far, although I followed their protocol to the word.
I have digested my viral genomic DNA with HindIII and used the appropriate kit (Copy Control HindIII) but I could not even get a single colony on my plates.
I have tried everything, from slightly changing ligation conditions to re-circularizing the vector and cutting it with my fresh HindIII enzyme.
If there's anyone out there using the same kit with some useful advice, please let me know!!!
I'm consructing BAC library for egyptian wheat
I've a problem with using the same kit I bought it , the kit you used didn't give you any clone but in my case it gave me only white clonies and when I tested them for characterization I discovered that the clones didn't carry any fragment although it is white and I asked the company for the expire date of the kit they didn't reply
what did you do in ur case to bypass the problem
abandon the kit altogether as it is horrible!!!
What I have done is shotgun cloning (products of about 500bp ) in a BAC vector we use internally. You can basically pick anything that worked for you or your colleagues earlier...
I got into a world of trouble with my boss trying to clone this way. He thought I did not know what I was doing and came short of calling me incompetent....