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blunt and sticky end clonning temperature - (Apr/27/2011 )

Hi there !

I have an exam tomorrow and i need to know something i cant understand. In blunt end cloning,optimal temperature is 22C and incubation time is 6 hours. But sticky end clonning, we use 16C and 12-16 hours. There is a big difference but why ? the numbers are not important my point is why is there such a difference like that ?
looking forward to your messages...

Fatih

-ofbaltaci-

ofbaltaci on Wed Apr 27 17:39:11 2011 said:


Hi there !

I have an exam tomorrow and i need to know something i cant understand. In blunt end cloning,optimal temperature is 22C and incubation time is 6 hours. But sticky end clonning, we use 16C and 12-16 hours. There is a big difference but why ? the numbers are not important my point is why is there such a difference like that ?
looking forward to your messages...

Fatih


Au contraire, the numbers are very improtant because it seems you have them swapped. Blunt ligations you do at lower temperature (4 or 16C) and longer ligation time (12-16h) than sticky ligations (1h, RT or 37c), because you want the ends of the fragments to move as little as possible, while giving the inefficient joining reaction of the two blunt ends as much time as possible.

-hematopoietry-

hematopoietry on Thu Apr 28 11:14:39 2011 said:


ofbaltaci on Wed Apr 27 17:39:11 2011 said:


Hi there !

I have an exam tomorrow and i need to know something i cant understand. In blunt end cloning,optimal temperature is 22C and incubation time is 6 hours. But sticky end clonning, we use 16C and 12-16 hours. There is a big difference but why ? the numbers are not important my point is why is there such a difference like that ?
looking forward to your messages...

Fatih


Au contraire, the numbers are very improtant because it seems you have them swapped. Blunt ligations you do at lower temperature (4 or 16C) and longer ligation time (12-16h) than sticky ligations (1h, RT or 37c), because you want the ends of the fragments to move as little as possible, while giving the inefficient joining reaction of the two blunt ends as much time as possible.


The discrepancy also is a trade off between optimal temperature for ligase (some 37, promega says 25) and the annealing of the ends between your vector and fragment inserted, which from memory is about 14 (won't take that number to heart though).

-Abousy-