Ligation problem: Phosphotasing Sac I ends.

brett brett at BORCIM.WUSTL.EDU
Wed Oct 16 12:38:04 EST 1996


>I'm having difficulty cloning a 7 kb Sac I fragment into pUC 19.  One
>problem has been the phosphotasing of pUC 19/SacI.   I phosphatased the
>vector twice with shrimp alkaline phosphotase.  Comparing transformations
>of vector without ligase and vector with ligase,  I get thousands of blue
>colonies for the vector with ligase.  Self ligation of the vector in the
>absence of insert has also been confirmed by gel electrophoresis.  
>Apparently the 5' *recessed* phosphates are not being removed completely. 
>Any suggestions?
>
>Pat M.

Yeah, those recessed phosphates can be tricky. I'd use 2-3x excess CIAP(!) at
50C for 1 hr, then clean up the vector. Your background should be very low.


Brett Lindenbach
    
Program in Immunology                              
Washington University - St Louis                  
brett at borcim.wustl.edu                             

"I own my own pet virus. I get to pet and name her." - Cobain




More information about the Methods mailing list