Insert is double the size than vector - (Nov/01/2006 )
I am trying to clone a gene to PUC19 and the insert is double the size of PUC19. I want to clone in SalI site. I dephosphorilated PUC after SalI digestion and and ligated with insert which was cloned in another plasmid in SalI site. All the colonies which I got were self ligated PUC19 without insert. I think it is the problem of dephosphorylation. I used fermentas CIP and incubated for 1 hr. Could any one of you give some suggestions to inprove my cloning? I am under pressure. Thanks in advance.
dephosphorylate the vector properly and do a 1:1 ligation.
today i just got my colonies where the vector is 3kb and insert 8.1 kb. I cipped the vector with antarctic CIP from Neb and ligated with a ration vector/insert 1:3 (1 to 1 didt work for me)
ligase promega ON at 4C
I got self ligation but surely less than without cippping
Personally I would avoid digesting my vector with SalI. Call it superstition but I have had several very trying experiences with that restriction enzyme. I think that enzyme does something funny to the ends. Could you use a different enzyme?
Now aside from my personal belief, I am concern about the CIP step. How many units of CIP are you using and how much DNA is being dephosphorylated? 1hr is quite a long time.
I use the following rule
0.1U of CIP will dephosphorylate 1pmol of DNA in 1hr
It should be noted that CIP is not a clean enzyme, over dephosphorylation with Calf Alkaline Phosphotase will damage the end of your DNA. Perhaps that is the cause?
Also how large is the volume of your ligation reaction? As your insert is rather large, the self ligation reaction is favoured. A concentrated vector-insert ligation mix can better push the ligation reaction towards pickup of insert.
Make sure you clean up your DNA after treating with the phosphatase. The CIP can not be inactivated easily, so usually you need to some othe rkind of clena-up precedure of your choice.
Our dephosphorylation takes a total of 1 hr, but in various stages. After digesting w/ the REs (20uL total volume) we add
5uL AP Buffer
5uL CIP diluted 1:100
(Final volumeL 50uL)
Digest at 37C for 15 min, Digest at 56C for 15 min.
Add additional 5uL diltuted CIP
Digest at 37C for 15 min, Digest at 56C for 15min.
Heat inactivate at 65C for 15 min.
Usually we then just run 5uL on a gel to test the results, but you could also do a Wizard prep on the sample if you are concerned about having CIP or any RE left in the mix
Thank u all, I really got a good idea from all your reply.