Subcloning transgene from a BAC clone - Subcloning transgene from a BAC clone (Jun/26/2003 )
Our lab is trying to develop a transgene construct for a knock-out murine model. I tried to isolate our gene approx. 10 KB (its rather small) using long-accurrate PCR. The genomic sequence for another mouse strain C57BL/6 is listed at NCBI for our gene of interest which I used to design primers for 129SV genomic mouse DNA. I had minimal success after trying multiple annealing temperatures and optimizations. I received funding to utilize an outside source to screen a 129SV BAC library for our gene of interest for our desired clone. My question is what to do with the BAC clone once I receive it. Should I try again to PCR out this gene from the clone? I would like to avoid having to digest and sequence the whole clone. Any information would be helpful since I am new to this.
To construct a subclone library from BAC. Perhaps u can construct a 15Kb library using phage vector.Is there any feature of ur gene for screening? If there's not,
u should disign a probe and do southen blot.Indeed I was trying to construct a 10Kb library using pUC18 as vector.Though it's not easy,but for BAC, it should be very easy since u need only a few clones.