20 replies to this topic

[DNA]

Dear All,

I have a DNA construct which is already cloned in a vector   pCMV6-XL4 and I want to remove it from this vector and clone in to
another vector PCDNA1.1/AMP. Can some on eplease elaborate on the steps to accomplish this task please. I know it sounds more like a homework question but my background is not molecular biology or biotechnology and in my lab there i sreally no one who can guide me.
I would be very thankful if some one on thi sforum can help me out.

Thanks

Edited by DNA, 28 August 2010 - 03:36 AM.

[pito]

do you know anythiong about this vector? restriction sites etc?

[HomeBrew]

There is a map of pCMV6-XL4 here, and one of PCDNA1.1/AMP here.

The basic steps are to release your insert from its current vector using appropriate restriction enzyme(s), purify the released fragment from the pCMV6-XL4 backbone, and ligate the insert into appropriately digested PCDNA1.1/AMP, and recover the clones via chemical transformation or electroporation of a suitable bacterial host, selecting for successful transformants using the ampicillin resistance marker carried by PCDNA1.1/AMP.

[ranvi]

Since you dont have Mol.biology back ground you should check every step with someone in your lab or at your dept, becos sometimes it might take several months to figure out the problem for freshers.

Check for the same restriction sites like if your DNA is cloned with pCMV6-XL4 at  ECORI , NotI  and if you have the same Restriction site PCDNA1.1/AMP

all you have to do is digest with these enzyme which will open  the pCMV6-XL4, and your Dna will drop out of this vector. Cut the   PCDNA1.1/AMP in similar fashion which will open it too. after that gel purify these.

take the dna alone from the gel and purify it, also after enzyme restriction purify the PCDNA then run it on the gel to make sure they are clean,  you can check the m.wt. On the gel you should see only a single band, if you have many bands then not enough cleaning

Then go for the ligation
either ligafast promega or NEB overnight ligation both works
after ligation do the transformation, check the control (which should not give colonies) and your ligation, then screen the colonies for the insertion of dna, you can check by doing the same enzyme digestion


It will be more clear if you talk to anyone personally

Edited by ranvi, 28 August 2010 - 01:17 PM.

[DNA]

HomeBrew, on 28 August 2010 - 07:01 AM, said:

There is a map of pCMV6-XL4 here, and one of PCDNA1.1/AMP here.

The basic steps are to release your insert from its current vector using appropriate restriction enzyme(s), purify the released fragment from the pCMV6-XL4 backbone, and ligate the insert into appropriately digested PCDNA1.1/AMP, and recover the clones via chemical transformation or electroporation of a suitable bacterial host, selecting for successful transformants using the ampicillin resistance marker carried by PCDNA1.1/AMP.

You have rightly pointed out the maps of tehvectors but I just checked the invitrogen website and it says pcDNA1.1/amp is no longer available, so now I will be using the pcDNA3+ vector. I know the rest of the steps but I just dont know which enzymes I will be using to release the insert from the pcmvx-Xl4 vector and then which enzyme i will be using to digest the pcDNA3+ vector to ligate my insert. Can you please guide or suggest me where can I find these informations. I have bought the true clone from Origene.

[DNA]

ranvi, on 28 August 2010 - 01:17 PM, said:

Since you dont have Mol.biology back ground you should check every step with someone in your lab or at your dept, becos sometimes it might take several months to figure out the problem for freshers.

I will try to do so
Check for the same restriction sites like if your DNA is cloned with pCMV6-XL4 at  ECORI , NotI  and if you have the same Restriction site PCDNA1.1/AMP
[b]Here is my main problem, I dont know how to find the restrition sites for the pcmv6-XL4 and how to check what restriction sites are present in pcDNA3+ (I will use this one, as I checked and pcDNA1.1/amp is no longer available from invitrogen.

all you have to do is digest with these enzyme which will open  the pCMV6-XL4, and your Dna will drop out of this vector. Cut the   PCDNA1.1/AMP in similar fashion which will open it too. after that gel purify these.

take the dna alone from the gel and purify it, also after enzyme restriction purify the PCDNA then run it on the gel to make sure they are clean,  you can check the m.wt. On the gel you should see only a single band, if you have many bands then not enough cleaning

Then go for the ligation
either ligafast promega or NEB overnight ligation both works
after ligation do the transformation, check the control (which should not give colonies) and your ligation, then screen the colonies for the insertion of dna, you can check by doing the same enzyme digestion


It will be more clear if you talk to anyone personally

The rest of the process liek gel extraction, transformation and selection, I have been doing these proceduresregularly so I know about them and I can do them

Thanks for you help

[pito]

Where is the insert in your vector? You need to tell this so we can tell you wich restriction enzyme to use to get it out of the vector.

Its like ranvi allready told you: you need to know more about the insert.

In general its rather easy: you simple take a restriction enzyme (or more) to cut out the DNA part you want , clean this up. Then cut the new vector with the same or another (complementary) restriction enzyme , clean this up too. And then: you add both together... et voila..


Use the maps Homebrew gave you to see wich restriction enzymes you can use...

But you do know how the insert in the original vector was created , right?

[DNA]

pito, on 29 August 2010 - 06:00 AM, said:

Where is the insert in your vector? You need to tell this so we can tell you wich restriction enzyme to use to get it out of the vector.

Its like ranvi allready told you: you need to know more about the insert.

In general its rather easy: you simple take a restriction enzyme (or more) to cut out the DNA part you want , clean this up. Then cut the new vector with the same or another (complementary) restriction enzyme , clean this up too. And then: you add both together... et voila..


Use the maps Homebrew gave you to see wich restriction enzymes you can use...

But you do know how the insert in the original vector was created , right?

Ok, I got some information now.
Actually I have orderdred true clone from origene , here i steh product

http://www.origene.c...7&sku=SC119669.

It says the restriction site is Not1-Not1 and i can release the insert using Not1.
Now once I release it, I want to put in pcDNA3.1(+) I have attached the pDF file which has given the maps of pcDNA3 vector.
Now since I know that I can release the insert by using Not1, I will gel purify it???Am I right?
So in the next step which enzyme/s will I use to open/digest the pcDNA3.1 (+) vector to put my DNA construct there.

Thanks a lot for help. I really appreciate.

Attached Files

•  pcDNA3.pdf   250.72K   11728 downloads

[HomeBrew]

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.

[Chakchel]

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.

[DNA]

Chakchel, on 01 September 2010 - 12:32 AM, 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?

[pito]

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...

[DNA]

HomeBrew, on 31 August 2010 - 06:38 PM, 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?

Edited by DNA, 01 September 2010 - 03:58 AM.

[Chakchel]

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...

[DNA]

Chakchel, on 01 September 2010 - 04:23 AM, 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?