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Oligo annealing - phosphorylation necessary? - (Aug/02/2009 )

Hello all,

I was just wondering if it makes any difference whether you phosphorylate the oligos before annealing them or after annealing them, if you are trying to ligate oligos in a blunt vector site?

Best,
TC

-T C-

T C on Aug 2 2009, 10:39 PM said:

Hello all,

I was just wondering if it makes any difference whether you phosphorylate the oligos before annealing them or after annealing them, if you are trying to ligate oligos in a blunt vector site?

Best,
TC


As long as one of the two ends for ligation is phosphorylated (blunt vector end), you don't need to phosphorylate the oligos. In fact, if you do phosphorylate oligos, there are chances that you may end up getting concatemers of oligos ligated to your vector.

So, just use the annealed oligos directly for ligation.

-cellcounter-

cellcounter on Aug 3 2009, 10:44 PM said:

T C on Aug 2 2009, 10:39 PM said:

Hello all,

I was just wondering if it makes any difference whether you phosphorylate the oligos before annealing them or after annealing them, if you are trying to ligate oligos in a blunt vector site?

Best,
TC


As long as one of the two ends for ligation is phosphorylated (blunt vector end), you don't need to phosphorylate the oligos. In fact, if you do phosphorylate oligos, there are chances that you may end up getting concatemers of oligos ligated to your vector.

So, just use the annealed oligos directly for ligation.


Just a question here.

This would also mean that there is a chance of concatamers of vectors (which is way much better than concatamers of inserts of course) and they can self ligate together. Upon transformation, will they still be able to have antibiotic resistance?

-jiajia1987-

jiajia1987 on Aug 3 2009, 05:11 PM said:

cellcounter on Aug 3 2009, 10:44 PM said:

T C on Aug 2 2009, 10:39 PM said:

Hello all,

I was just wondering if it makes any difference whether you phosphorylate the oligos before annealing them or after annealing them, if you are trying to ligate oligos in a blunt vector site?

Best,
TC


As long as one of the two ends for ligation is phosphorylated (blunt vector end), you don't need to phosphorylate the oligos. In fact, if you do phosphorylate oligos, there are chances that you may end up getting concatemers of oligos ligated to your vector.

So, just use the annealed oligos directly for ligation.


Just a question here.

This would also mean that there is a chance of concatamers of vectors (which is way much better than concatamers of inserts of course) and they can self ligate together. Upon transformation, will they still be able to have antibiotic resistance?


yes, if the ends of the vector are compatible (complementary overhangs or blunt end), leaving said ends phosphorylated will increase the number of colonies recovered which contain self-ligated vector. Generally if the end of the vector are incompatible (often cut by different restriction enzymes), then the vector will not religate. Probables arise from incomplete digestions, where the vector is only cut once, leaving it with compatible ends.

Vector concatermization is possible but these molecules have a low probability of being recovered. Plasmid with 2 origins of replication are not able to propagate well.

-perneseblue-

Hey

I need to avoid religation of vector so CIP treatment of vector is necessary and oligos must be phosphorylated, the point is before or after annealing.

Personally, I don't think it makes a difference but was wondering if anyone has had any experience with this.

Best,
TC

cellcounter on Aug 3 2009, 09:14 PM said:

T C on Aug 2 2009, 10:39 PM said:

Hello all,

I was just wondering if it makes any difference whether you phosphorylate the oligos before annealing them or after annealing them, if you are trying to ligate oligos in a blunt vector site?

Best,
TC


As long as one of the two ends for ligation is phosphorylated (blunt vector end), you don't need to phosphorylate the oligos. In fact, if you do phosphorylate oligos, there are chances that you may end up getting concatemers of oligos ligated to your vector.

So, just use the annealed oligos directly for ligation.

-T C-