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Large vector self-ligation in presence of insert (1:1)? - (Jun/24/2008 )

I am trying to reduce the size of a very large plasmid by digesting out a fragment that I don't need. Due to the large size of the resulting vector (11 kb) I want to recircularize the large fragment (containing kanR) in the presence of the digested insert (2 kb). Will blunt-end ligation in a 1:1 mixture of vector:insert result in enough self-ligation products for me to find one using colony PCR? Would high DNA concentration in the ligation mix favor self-ligation?

-jeffl-

QUOTE (jeffl @ Jun 24 2008, 10:40 AM)
I am trying to reduce the size of a very large plasmid by digesting out a fragment that I don't need. Due to the large size of the resulting vector (11 kb) I want to recircularize the large fragment (containing kanR) in the presence of the digested insert (2 kb). Will blunt-end ligation in a 1:1 mixture of vector:insert result in enough self-ligation products for me to find one using colony PCR? Would high DNA concentration in the ligation mix favor self-ligation?

You will be fine. Most of the colonies will be self ligated due to molecular ratio and overall vector size. However, some of them may be undigested ones if you do not purify the fragments.

You may also think of adding a second enzyme that cuts the 2kb insert, thereby reducing any chance of its getting cloned. Edit: Using two enzymes will also make it almost sure that you have no undigested plasmid left.

-cellcounter-

QUOTE (cellcounter @ Jun 24 2008, 02:48 PM)
QUOTE (jeffl @ Jun 24 2008, 10:40 AM)
I am trying to reduce the size of a very large plasmid by digesting out a fragment that I don't need. Due to the large size of the resulting vector (11 kb) I want to recircularize the large fragment (containing kanR) in the presence of the digested insert (2 kb). Will blunt-end ligation in a 1:1 mixture of vector:insert result in enough self-ligation products for me to find one using colony PCR? Would high DNA concentration in the ligation mix favor self-ligation?

You will be fine. Most of the colonies will be self ligated due to molecular ratio and overall vector size. However, some of them may be undigested ones if you do not purify the fragments.

You may also think of adding a second enzyme that cuts the 2kb insert, thereby reducing any chance of its getting cloned. Edit: Using two enzymes will also make it almost sure that you have no undigested plasmid left.


Thanks again cellcounter. I should have mentioned that I'm double digesting out the fragment then using klenow to blunt-end the overhangs. As a result, cutting the 2kb insert would require another step after deactivating klenow. I still like your idea, though there doesn't appear to be a convenient unique RE site in the 2kb fragment that would produce sticky ends.

-transient-

QUOTE (transient @ Jun 24 2008, 07:35 PM)
QUOTE (cellcounter @ Jun 24 2008, 02:48 PM)
QUOTE (jeffl @ Jun 24 2008, 10:40 AM)
I am trying to reduce the size of a very large plasmid by digesting out a fragment that I don't need. Due to the large size of the resulting vector (11 kb) I want to recircularize the large fragment (containing kanR) in the presence of the digested insert (2 kb). Will blunt-end ligation in a 1:1 mixture of vector:insert result in enough self-ligation products for me to find one using colony PCR? Would high DNA concentration in the ligation mix favor self-ligation?

You will be fine. Most of the colonies will be self ligated due to molecular ratio and overall vector size. However, some of them may be undigested ones if you do not purify the fragments.

You may also think of adding a second enzyme that cuts the 2kb insert, thereby reducing any chance of its getting cloned. Edit: Using two enzymes will also make it almost sure that you have no undigested plasmid left.


Thanks again cellcounter. I should have mentioned that I'm double digesting out the fragment then using klenow to blunt-end the overhangs. As a result, cutting the 2kb insert would require another step after deactivating klenow. I still like your idea, though there doesn't appear to be a convenient unique RE site in the 2kb fragment that would produce sticky ends.

Sorry, I missed it!
If you are using blunt ligation, there is no chance (even in hell), that the insert would be ligated. Having such ligation work would be a dream to many. laugh.gif
So, just go ahead!

-cellcounter-