In article <199610161736.MAA06281 at wustl.edu>, brett at BORCIM.WUSTL.EDU (brett) wrote:
>>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
>I use CIP and when phosphotasing blunt and recessed ends, I incubate the
reaction with 1 unit of ez. at 37 for 30 min , add another unit and continue
incubation at 55 for 1 hr. This works for me
Hope this helps
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Karthikeyan Ramachandran Ph.D
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School of Biological and Chemical Sciences
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