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Ligation problem: Phosphotasing Sac I ends.

Karthi Ramachandran karthi at deakin.edu.au
Thu Oct 17 18:48:34 EST 1996

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

Karthikeyan Ramachandran Ph.D
Research Fellow, Rm. No. SA136
School of Biological and Chemical Sciences
Faculty of Science and Technology	Phone (Off) (052) 271 406
Deakin University			Phone (Res) (052) 471 806
Geelong, Victoria			Fax ()52) 272 022
Australia 3217				e-mail karthi at deakin.edu.au
Home page				http://www.deakin.edu.au/~karthi/

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