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How to ligate ds-oligos into a vector?


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23 replies to this topic

#16 vesko_baev

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Posted 23 March 2005 - 07:02 AM

Hi,
I have a 25nt ds oligo insert and about 4kb 2digested vector. What rato is good to make? Can I use for the ligation, the phosphorylation reaction solution right away, or I have to clean the phosph. oligo?

Thanks

#17 Landene

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Posted 23 November 2009 - 01:46 AM

Gel extracting the oligos reduces the amount of shorter oligo product in your sample and thus increases the chances for a successful ligation.

Regards

Landene
Surendettement

#18 William Parkar

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Posted 21 April 2010 - 10:18 PM

If you are still having trouble, gel purify your oligos first on a polyacrylamide gel, then anneal the two oligos and phosphorylate them. Dephosphorylate your digested vector and gel extract that as well. Then set up your ligation in 10uL. I've done this many times and it will even work with the quick ligation kits. Good luck.


http://www.flashpapers.com

#19 chemy333

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Posted 28 November 2010 - 12:37 PM

There is a method called Ligation assisted by nucleases (have a look at this paper in Nature protocols: http://www.nature.co...t.2007.325.html)

Basically, you design your primers to have overhangs complementary to the restriction sites, but such that once they are ligated in, the restriction sites in the original vector are lost.

You run your ligation by mixing your circular vector + annealed primers + restriction enzyme + T4 DNA ligase in one single tube. If the vector re-circularizes, it gets cut again by the restriction enzyme. If the primers get inserted, the restriction sites are lost, and the product remains circular. The method works best with enzymes leaving longer overhangs (I have successfully used it in the past with XhoI/SbfI sites).

vector:
...CTCGAG
...GAGCTC

after cutting
...C____
...GAGCT

oligo overhang
TCGAH..... (H=not G)
____D..... (D=not C)

after ligation (restriction site lost)
...CTCGAH...
...GAGCTD...


In the past, I got >10x efficiency over self-ligation. Oligos should be non-phosphorylated, and pre-annealed (5' @95C, then slow cooling to 4C or similar).

Edited by chemy333, 28 November 2010 - 12:41 PM.


#20 ampelo

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Posted 07 July 2011 - 12:25 PM

Hi,

I read one of your query regarding annealing two primers and ligating it with a vector. I wanted to know if that worked for you. I am also interested in following similar strategy. I want to insert customized polylinker into my binary vector. Would you mind sharing your protocol. I will appreciate your help!

Ampelo!

Hi
I read your replies to enthusiast and I had similar problems and my primers also have the restriction sites at the end of them of the same enzyme to be later ligated in the sinly cut vector. I tried dephos my vector and ordered my primers 5' phosphorylated. I annealed the primers by heating 94 C fro 1 min and at room temp for 1hr. and also as suggested by some one I annealed them by placing in a water bath 70C for 1-2hr and bringing temp down slowly for a whole day say 10 each hr. I still get as many colonies on my control plate (no insert) after ligation. I even tried cutting the dephosphorylated digested vector off the gel and purify it. I did however never gel purify my insert (the annealed!! primers). How would I do that? Do I just let anneal and run on gel and purify or......???
I would be grateful if you could suggest why I am getting similar no. of colonies on my control (no insert) plate as my ligation plates?
Thank you in advance



#21 ampelo

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Posted 07 July 2011 - 12:33 PM

Its really a cool idea! will try getting the article. Thanks for spreading the knowledge! thats how science progresses!

There is a method called Ligation assisted by nucleases (have a look at this paper in Nature protocols: http://www.nature.co...t.2007.325.html)

Basically, you design your primers to have overhangs complementary to the restriction sites, but such that once they are ligated in, the restriction sites in the original vector are lost.

You run your ligation by mixing your circular vector + annealed primers + restriction enzyme + T4 DNA ligase in one single tube. If the vector re-circularizes, it gets cut again by the restriction enzyme. If the primers get inserted, the restriction sites are lost, and the product remains circular. The method works best with enzymes leaving longer overhangs (I have successfully used it in the past with XhoI/SbfI sites).

vector:
...CTCGAG
...GAGCTC

after cutting
...C____
...GAGCT

oligo overhang
TCGAH..... (H=not G)
____D..... (D=not C)

after ligation (restriction site lost)
...CTCGAH...
...GAGCTD...


In the past, I got >10x efficiency over self-ligation. Oligos should be non-phosphorylated, and pre-annealed (5' @95C, then slow cooling to 4C or similar).



#22 ampelo

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Posted 07 July 2011 - 01:05 PM

Would you mind giving me your protocol? I want to try it.


Its really a cool idea! will try getting the article. Thanks for spreading the knowledge! thats how science progresses!


There is a method called Ligation assisted by nucleases (have a look at this paper in Nature protocols: http://www.nature.co...t.2007.325.html)

Basically, you design your primers to have overhangs complementary to the restriction sites, but such that once they are ligated in, the restriction sites in the original vector are lost.

You run your ligation by mixing your circular vector + annealed primers + restriction enzyme + T4 DNA ligase in one single tube. If the vector re-circularizes, it gets cut again by the restriction enzyme. If the primers get inserted, the restriction sites are lost, and the product remains circular. The method works best with enzymes leaving longer overhangs (I have successfully used it in the past with XhoI/SbfI sites).

vector:
...CTCGAG
...GAGCTC

after cutting
...C____
...GAGCT

oligo overhang
TCGAH..... (H=not G)
____D..... (D=not C)

after ligation (restriction site lost)
...CTCGAH...
...GAGCTD...


In the past, I got >10x efficiency over self-ligation. Oligos should be non-phosphorylated, and pre-annealed (5' @95C, then slow cooling to 4C or similar).



#23 StephanK82

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Posted 09 September 2011 - 04:09 AM

Hi!

There is also another protocol from Invitrogen. I use that regularly to anneal oligos and it always works. However, I order the primers as HPLC purified oligos. Usually, 1 out of 15 so far had a mutation, annealing and cloning always worked... I also used different sizes of oligos (30 to 100nt) always with the same protocol. So far, I did not see a difference in efficiency.

The link to the protocol is here:

https://rnaidesigner...ss/ds_oligo.pdf

cheers,
Stephan

#24 allynspear

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Posted 21 September 2011 - 11:45 AM

A properly dephosphorylated vector should give Zero colonies on from a vector only ligation/transformation. I don't know what phosphatase you are using or how you are preparing your vector, but in my experience, here is the best protocol:

1) Digest 10 ug of vector DNA
2) Heat inactivate the Restriction enzyme if possible
3) Gel purify linearized vector DNA on an agarose gel
4) Dephosphorylate your gel purified vector using either Shrip Alkaline Phosphatase (SAP) or Antarctic Phosphatase,
5) Heat inactivate the phosphatase
6) Quantitate your vector concentration by agarose gel

Using this protocol, I routinely get zero colonies on a vector only ligation. For annealing two oligonucleotides, I routinely use a PCR machine and I perform the annealing in 1X PCR buffer. If you have a PCR machine that lets you control the cooling rate, then heat to 94 degrees and slow the cooling rate as much as you can and cool to 4 degrees. If not, just program a series of 10 degree drops from 94 to 4, for 5 minutes each (94, 5min; 84, 5min; 74, min; etc).

You could possibly be having self annealing problems with your primers if they are too complimentary, so you can run a program like oligoanalyzer to check it or post the primer sequences and someone could let you know if there is a problem. Do not waste time purifying your annealed oligos on a polyacrylamide gel, since that is way too much work for a simple dsOligo ligation.

Best of Luck.

Edited by allynspear, 21 September 2011 - 11:48 AM.





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