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plasmid DNA and restrictions enzyme - (Jun/06/2006 )

hmm.. why is it that alkaline phosphatase is added to treat restriction enzyme digested plasmid DNA before ligation with unsert DNa?

is it that the alkaline phophatase will break the hydrogen bonds in the plasmid DNA and allows the plamid DNA to be separated and allows ligation? but isnt the plasmid DNA already cut earlier by the restriction enyme, or is it that the plamid DNA will rejoin back after the pH is normal?


when you're doing a restriction enzyme reaction, you break the phosphodiester bond between 3'O of the first base, an the phosphate.
So you obtain as ends a 3' OH and a 5' phosphate, which is actually the template required for a ligation. As you know, restriction cloning is done generally with 2 different enzymes, and so the vector may not religate itself. For more security, you can remove the 5' phosphate of the vector by the phosphatase.


what do u mean by 2 differnet enzyme? ligase or the restriction enzyme?


all right, let's back up a step

when you cut a vector for cloning, oftentimes you will use two different restriction enzymes in order to control the direction of the insert

as Fred said, if you only use one, you need to phosphatase in order to remove the phosphate hanging off the 5' end of the DNA strand. if you don't, the vector is more likely to simply ligate back to itself during the ligation reaction. if you remove the phosphate, this makes this re-ligation highly unlikely and increases your chances of getting the correct insert to ligate into the vector

even if you clone with two restriction enzymes, often it is wise to dephosphorylate the vector...if you are using two sites in the multiple-cloning-site, it can be difficult to determine for certain if both enzymes have cut properly...this leads to the same situation as is the single-enzyme example, and you don't want most of your transformants to be re-ligated vector. screening tons of colonies is a real pain

if you look at the name of the enzyme, often you can get a clue into what function it performs...'phosphatase' removes overhanging phosphates, you see?


oh icic..this makes it a whole lots easier.. but y isnt phosphatase added to the DNA.. wouldnt it religate back to itself as well?

btw how do i get the warning off??


So you start with your cut vector. The ends look like this:

vector--OH   P--vector
vector--P   OH--vector

Then you add CIP to the DNA, which removes the phosphates:

vector--OH   --vector
vector--   OH--vector

Now, the ligase cannot rejoin those ends, because it needs to link a P with an OH, and you've removed the phosphates. So your vector cannot close back into a circle.

Now add your (non-dephosporylated) insert into a mix with your dephosphrylated vector and the ligase, and you get:

vector--OH  P--insert--OH   --vector
vector--  OH--insert--P  OH--vector

The ligase can join the vector to your insert, once on each strand, leaving two single stranded breaks, which are repaired when the plasmid is replicated.

-HomeBrew- then i truly understand the restiction digestion and ligation process..thnx man.. biggrin.gif
guess that i have to be more attentive in clss..