How to ligate ds-oligos into a vector? - (Aug/25/2004 )
Its really a cool idea! will try getting the article. Thanks for spreading the knowledge! thats how science progresses!
chemy333 on Sun Nov 28 20:37:08 2010 said:
There is a method called Ligation assisted by nucleases (have a look at this paper in Nature protocols: https://www.nature.com/nprot/journal/v2/n9/full/nprot.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).
Would you mind giving me your protocol? I want to try it.
ampelo on Thu Jul 7 20:33:18 2011 said:
Its really a cool idea! will try getting the article. Thanks for spreading the knowledge! thats how science progresses!
chemy333 on Sun Nov 28 20:37:08 2010 said:
There is a method called Ligation assisted by nucleases (have a look at this paper in Nature protocols: https://www.nature.com/nprot/journal/v2/n9/full/nprot.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).
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.invitrogen.com/rnaiexpress/ds_oligo.pdf
cheers,
Stephan
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.