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ligation problem


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#1 deespike

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Posted 26 July 2010 - 08:01 AM

HI ALL

I am currently working on a cloning of a 2.8 kb insert into a 12kb vector and one end being sticky and the other being blunt . Now i am really confused about how i deal with it. I use neb ligase with 400cohesive ligation units / ul. How much neb ligase is really required for blunt ligation?In the neb site its mentioned as adding concentrated ligase but whats the range of it, doesnt mention.

Can anyone please help?

#2 molstudent

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Posted 26 July 2010 - 02:35 PM

HI ALL

I am currently working on a cloning of a 2.8 kb insert into a 12kb vector and one end being sticky and the other being blunt . Now i am really confused about how i deal with it. I use neb ligase with 400cohesive ligation units / ul. How much neb ligase is really required for blunt ligation?In the neb site its mentioned as adding concentrated ligase but whats the range of it, doesnt mention.

Can anyone please help?


I always use 1ul of ligase. I did a ligation with one sticky end and one blunt end too and it worked fine.

#3 tfitzwater

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Posted 27 July 2010 - 07:25 AM

One Weiss unit corresponds to 67 cohesive-end units as defined by New England Biolabs.
400 NEB Units = 6 Weiss Units.
I use 6 NEB units for a sticky end ligation using 1 ug of vector, and 67 NEB Units for blunt end ligations overnight at 14C.
NEB's Quick ligation protocol uses two million NEB Ligase Units/mL = 30,000 Weiss Units/mL or 30 Weiss Units/L.
NEB T4 DNA ligase diluted to 6 Units/uL or 67 Units/uL in NEB Ligase Storage Buffer is good for more than 1 year at -20C. This will really stretch your lab's resources if you don't have a lot of money.

#4 deespike

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Posted 27 July 2010 - 09:15 AM

Thanks for your reply this helps . I have diluted the enzyme as 200cohesive ligation units /ul and I used 2 ul for ligation as i was scared about the blunt part. i am hoping for good result.

But i have a doubt regarding the temperature 14C . Generally its advised to perform blunt ligation at room temperature , low temperature will work?




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