Ligation problem: Phosphotasing Sac I ends.
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.
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.
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
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