How to clone DNA (already cloned in a vector) in to another vector - please help (Aug/28/2010 )
Chakchel on Wed Sep 1 08:32:15 2010 said:
Another important thing you should check before you try to cut your insert out: Is the the restriction enzyme you chose (NotI) cutting within your insert's sequence? Hopefully not.
How to check that? Neb Cutter? I did that but it says its not cutting.
However, currently we have the this insert in another old vector pcDM8 and we use Not1 to linearize/digest the plasmid DNA before invitro transcription. Does that mean Not1 is cutting within my insert or it is just cutting the vector pcDM8?
Its most likely just cutting the vector.
You said yourself that you checked the insert if it contained a Not1 restriction place and it doenst... so its ok.
You can most likely do what Homebrew stated.
Its simple: you need to get the insert out of the vector, you can use Not1 for that (knowing that Not1 doenst cut into your insert) and then you need to open the new vector, wich can be done with Not1 also...
HomeBrew on Wed Sep 1 02:38:19 2010 said:
There's a NotI site in the multiple cloning region of pcDNA3.1 -- why not use that? Gel purify both your NotI-digested insert and your NotI-digested and CIP-treated pcDNA3.1 vector, and ligate them together.
Thanks I got most of the concept now... But what is this CIP?
CIP is calf intestinal phosphatase.
You use it to dephosphorylate your vector after cutting. Otherwise religation of your vector may occur, and you get lots of colonies only containing the empty vector.
Instead of CIP you also can dephosphorylate your vector using SAP (Shrimp alkaline phosphatase) or Antarctic phosphatase...
Chakchel on Wed Sep 1 12:23:17 2010 said:
CIP is calf intestinal phosphatase.
You use it to dephosphorylate your vector after cutting. Otherwise religation of your vector may occur, and you get lots of colonies only containing the empty vector.
Instead of CIP you also can dephosphorylate your vector using SAP (Shrimp alkaline phosphatase) or Antarctic phosphatase...
Hmm Ok, Thanks for that.. So how to set up a reaction of teh vector with CIP? Is there any protocol from the manufacturer? Who supplies CIP?
DNA on Wed Sep 1 13:05:41 2010 said:
So how to set up a reaction of teh vector with CIP? Is there any protocol from the manufacturer? Who supplies CIP?
The company from which you're buying your restriction enzymes very likely carries some form of alkaline phosphatase, be it calf, shrimp, and/or antarctic. A protocol will be supplied by the manufacturer.
HomeBrew on Wed Sep 1 13:30:10 2010 said:
DNA on Wed Sep 1 13:05:41 2010 said:
So how to set up a reaction of teh vector with CIP? Is there any protocol from the manufacturer? Who supplies CIP?
The company from which you're buying your restriction enzymes very likely carries some form of alkaline phosphatase, be it calf, shrimp, and/or antarctic. A protocol will be supplied by the manufacturer.
Thanks... We are buying our enzymes from Biolab... I just checked and they have the CIP.
DNA on Wed Sep 1 13:05:41 2010 said:
Chakchel on Wed Sep 1 12:23:17 2010 said:
CIP is calf intestinal phosphatase.
You use it to dephosphorylate your vector after cutting. Otherwise religation of your vector may occur, and you get lots of colonies only containing the empty vector.
Instead of CIP you also can dephosphorylate your vector using SAP (Shrimp alkaline phosphatase) or Antarctic phosphatase...
Hmm Ok, Thanks for that.. So how to set up a reaction of teh vector with CIP? Is there any protocol from the manufacturer? Who supplies CIP?
There are a lot of protocols out there.
(http://wheat.pw.usda.gov/~lazo/methods/lazo/dephos.html)
Check your lab.. maybe someone knows more about it, but its needed to do it, if not your vector will selsligate..
EDIT: didnt see the previous posts from you and homebrew.. So you allready know everything now.
Good luck.
DNA on Wed Sep 1 13:05:41 2010 said:
Chakchel on Wed Sep 1 12:23:17 2010 said:
CIP is calf intestinal phosphatase.
You use it to dephosphorylate your vector after cutting. Otherwise religation of your vector may occur, and you get lots of colonies only containing the empty vector.
Instead of CIP you also can dephosphorylate your vector using SAP (Shrimp alkaline phosphatase) or Antarctic phosphatase...
Hmm Ok, Thanks for that.. So how to set up a reaction of teh vector with CIP? Is there any protocol from the manufacturer? Who supplies CIP?
CIP is sold by most companies. NEB is an example. They do provide protocols along with their product. Visit their site for a look see.
You can probably get CIP from whomever you get your restriction enzymes from. We use Promega. It comes with a protocol.