Ligation problem: Phosphotasing Sac I ends.

[Karthi Ramachandran]

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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