Double-stranded oligo ligation - (May/30/2007 )

Friends, I am trying ligate a oligo ds(40bp) into pCDNA3.1 vector.
The oligos sense and antisense were denatured 94°C x 2min. After were cold down in room temperature.

The ds-oligo was cut with HindIII and EcoRI x 24hrs following the intructions by "new england biolabs" for cut of oligos with these enzyme.

Then the oligo cut was directly ligated to 4°C overnight en pCDNA3.1 cut with both enzymes and purified.

The transformation was in Ecoli JM109 (treated with RbCl)

The clones, however are not positive when cut with NheI or XbaI others enzyme present in the vector...

This method is Ok? what can the mistake?

Is neccesary desphosporilate the oligo after cut with HindIII and EcoRI?
How can do this without loss the oligo in the precipitaction (by size)?

Hope somebody can help me o give a better protocol.

Thanks

Jorge

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i do differently.

I order oligos which after annealing have little extra bases and restore the appropriate sites.
Then, i'm sure that the site is present and all oligos are like 100% digested.

Following that, i do kination and it's ok for ligation.
No problem with remaining restriction activity.

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Hi,
Did you purify your insert after restriction?
You could dephosphorylate your vector, not your insert, to prevent self-ligation. To check self-ligation of your plasmid, you colud set up a ligation without insert, which ideally should give no colonies after transformation.

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I do not, because I thinking the insert after restriction assay can lost then I do not purify.(the insert size is 40bp)

IS POSSIBLE AFTER CIAP TREATMENT, USE DIRECTLY THAT FOR LIGATION WITHOUT TO PURIFY?

I do my ligations to 4°C overnight.

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I dont understand very well... You order your oligos, each oligo carry the restriction site more 5 base at the end.
The same for the other oligo. Then I also have the sites restore and I then can do the restriction assay.

Example, AGACAGAATTCacacacacacacaca
EcoRI

like this??

or your order your oligos ok for the cohesive ends??

If you do kination DO you have problems with the concatemer-oligos?

THANKS a lot

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I think you have to purify your digested fragment. Otherwise the small DNA fragments that were removed by the digestion will still stick to both ends, making ligation impossible. Usually you gel extract your digested fragments, but I agree with you that you may lose quite a lot of DNA because of the small size of your insert. I' ve never used such a small insert so maybe anyone else has a suggestion.
As far as the phosphatase is concerned, you have to inactivate it before ligation, phenol-chlorofom extraction for example. We always use antartic phosphatase (NEB), which can be heat-inactivated in 20 minutes (saves a lot of time).

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I do it like fred_33:
Design the oligos so that when they anneal they produce overhanging ends. That way you don't need to do any restriction digestion.

I would probably not purify the digestion, because you'll lose nearly all of your DNA with such a small fragment. It sounds like you're getting self-ligation of the vector, so you should certainly dephosphorylate the vector (if you haven't already done so).

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NO...
what I do
oligo 1 : 5' XXXXXXXGAATT
oligo 2 : 3' XXXXXXXC

after annealing i will get the ecoRI site ready to clone
the kination of my oligos don't give me concatemers. But i check by sequencing.

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Fred:

I think you mean this:

oligo1: 5' XXXXXXXG
oligo2: 3' XXXXXXXCTTAA

EcoRI leaves a 5' overhang.

This will only ligate if oligo2 has a 5' phosphate, either added during synthesis, or with PNK.

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yep sorry
i prefer add the 5' phosphate myself because price of P-oligo is so expensive for our lab.
Also the procedure is quite efficent

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