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How do I perform self-ligations using click cloning?
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Author:  converrl [ Fri Nov 12, 2010 6:51 pm ]
Post subject:  How do I perform self-ligations using click cloning?

I want to perform sequence deletions following by a self-ligation in MacVector 11.1 --how do I do this?

thanks!

Author:  Chris [ Sat Nov 13, 2010 9:25 am ]
Post subject:  Re: How do I perform self-ligations using click cloning?

Do you mean to excise some part of a vector by digesting then ligating it?

- If so select two restriction sites in the MAP view that you want to ligate together (Click on one, then shift click on the next).
- Switch to the EDITOR view and select EDIT _ DELETE

The sequence between the two cut sites will be excised and ligated back together.

(please note that since you will not be using the ligation dialogue it is up to you to ensure that the ends are compatible).

I hope this helps.

Cheers
Chris

Author:  converrl [ Sat Nov 13, 2010 1:20 pm ]
Post subject:  Re: How do I perform self-ligations using click cloning?

I'm assuming that I would have to modify the ends myself (as in a fill-in or chew back with Klenow?)

Thanks!

Author:  macadmin [ Tue Nov 30, 2010 9:27 am ]
Post subject:  Re: How do I perform self-ligations using click cloning?

Hi converrl,

You could use the ligation dialogue to modify the ends if you prefer. However, to do so you will need to assemble the fragments in a new window. This is a quicker way to do so.

I hope this helps!

Author:  converrl [ Sat Dec 04, 2010 4:11 pm ]
Post subject:  Re: How do I perform self-ligations using click cloning?

How do I clear the click cloning clipboard of it's current source sequence ends?

Thanks!

Author:  converrl [ Sun Dec 05, 2010 4:05 pm ]
Post subject:  Re: How do I perform self-ligations using click cloning?

The self-ligation I'd like to perform is to cut at a single site, fill it in (Klenow) and close it back up...is there a way to do this other than to edit the sequence manually?

Thanks!

Author:  macadmin [ Wed Dec 08, 2010 12:47 pm ]
Post subject:  Re: How do I perform self-ligations using click cloning?

converrl wrote:
How do I clear the click cloning clipboard of it's current source sequence ends?

Thanks!


When you digest the next piece of sequence it will clear the cloning clipboard. Otherwise you can just use EDIT > COPY and that will remove it

Author:  macadmin [ Wed Dec 08, 2010 12:50 pm ]
Post subject:  Re: How do I perform self-ligations using click cloning?

converrl wrote:
The self-ligation I'd like to perform is to cut at a single site, fill it in (Klenow) and close it back up...is there a way to do this other than to edit the sequence manually?

I'm afraid that this is not possible at the moment. We will be introducing a fragment cloning clipboard in a future version. This will show the digested fragment in a viewer and allow you to modify it. For example in your case you would select the single site and the entire sequence would then become the fragment, which you could Klenow modify.

Author:  Chris [ Mon Dec 10, 2012 10:00 am ]
Post subject:  Re: How do I perform self-ligations using click cloning?

This is now possible with the new Cloning Clipboard in MacVector 12.7.


    select the enzyme recognition site you want to remove
    Click DIGEST

This linearizes the plasmid and places it on the Cloning Clipboard.

    Switch to the Cloning Clipboard and view your new fragment
    Drag one end to the other end
    Modify the ends to blunt them both


Now a new circular sequence will be created from your digested fragment.

The origin of your plasmid will have changed to the digested site, so you may want to rotate the sequence to show the original sequence.

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