Dephosphorylation of vector before ligation - Is it required? (Jan/26/2005 )
Hi all,
i am digesting my vector with 2 diffrent rest enzymes(Bgl II + Mlu I) yeilding diff sticky ends. after digesting do i have to proceed to dephosphorylation or can i skip it. in theory the sticky ends generated above would prevent self-ligation right?
i want to know is dephosphorylation is absloutely essential in such cases.
thx 
rajgene
In theory it's not absolutely necessary to dephosphorylate if you are using two REs with cohesive ends, but in fact, if the restriction efficiency of the enzymes is not 100%, you will get some molecules which have been cut only with one or the other enzyme, so they will religate if they are phosphorylated. One useful trick when cloning using two different sites is checking if you loose any site in the vector multiple cloning site (if any site is between the two REs you are using and not anywhere else in the vector). If this is the case, and this site is not found neither in your insert, you can digest your ligation with that enzyme and you will cut only your religated plasmids and not the ones with your insert, which will not have that site. So you will get only colonies with the insert.
Wow....wht a trick. badcell is quite good..
Yeah, that dirty little trick has spared me a lot of minipreps...
Digest the Ligation reaction. man, you are good. by the way, about the Miniprep, actually, how much DNA can you get from one miniprep? sometimes, i got very low concentration. is there any trick? thanks.
I usually get around 20-40 micrograms DNA from a 3-5 ml overnight culture, using qiagen columns or something of the sort. The quantity of DNA will vary depending if your plasmid is low-copy or high-copy. One good thing is not to overload the column, use 3-5 ml for a high-copy plasmid, or 10 ml for a low-copy. This is a case of less is more. Cheers!
I usually get around 20-40 micrograms DNA from a 3-5 ml overnight culture, using qiagen columns or something of the sort. The quantity of DNA will vary depending if your plasmid is low-copy or high-copy. One good thing is not to overload the column, use 3-5 ml for a high-copy plasmid, or 10 ml for a low-copy. This is a case of less is more. Cheers!
Hey Bad Cell the cloning master,
do u have ne idea of the percentage of false positives yeilded after transformation with and without rest.digestion after ligation? how many colonies shld i pick for colony pcr for confirming the clone on rest. digestion after ligation
Thx man!!
By the way, I'm a woman
Cheers!
Hi, Rajgene. You can do a control ligation with vector alone, digest it and transform it in parallel with the good one with the insert. If everything has gone alright you should get around 3-10 times more colonies in the ligation with the insert than in the control one. In that case you can pick 2-3 colonies, and if you are in luck they will all be good. A month ago I got only 3 colonies in the ligation plate and none in the vector alone plate. I picked the three colonies, and alll three of them were good
Cheers!
good Badcell. thx for ur thoughts. btw i am using roche Rapid DNA ligation kit. it says heat inactivation of ligase will decrease transformation efficiency.
so my Q is how do u go abt digestion after ligation. do i have to heat kill ligase before i put my enzyme or i can directly perform on the mixture.
please let me know some details on how to go abt it.
thx Lady.
cool control, badcell!! 
I will try this next time i do a ligation.
Doing a background control ligation is always important. Never do a ligation without negative controls, especially if you yourself cut the receiving vector.