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self-ligation Anneled oligos


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#1 pgarcia

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Posted 13 October 2011 - 09:01 AM

Hi everybody,

This is my first post and my english isn't too good.

I have a problem when I try to do a Self-ligation of anneled oligos.
I'll try to explain this:
I have a pair of oligos that I've anneled with a usual method ( heat at 95ºC and cool down at room temperature).
I ordered the oligos 5' phosphorilated and the oligos are head-tail cohesive.
When, after the oligo were anneled I try to do the self-ligation of the oligos to get multimers. I've ligated about 1ug of the anneled oligos with DNA ligase. I've run the ligation on Agarose gel and the oligos weren't ligated.

I wondered if I could get a protocol to do a self-ligation of oligos because I can't find anything about that.

Thanks very much,

#2 phage434

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Posted 13 October 2011 - 03:50 PM

You should be using plain T4 DNA ligation buffer (not quick ligation kits). You can heat kill this ligase reaction, which improves its gel behavior (otherwise you see DNA with ligase bound, making a mess on the gel). 80C for 20 minutes. Avoid all quick ligation buffers, which cannot be heat killed and will make a mess of your gel. What cohesive ends do you leave? What is your final goal?

#3 allynspear

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Posted 14 October 2011 - 06:17 AM

I completely agree with phage434, but I also have to say that looking for oligo ligation by agarose gel is very difficult. Your resolution is really low, unless your oligos are very large. But otherwise, looking for ligation of two or three 20 bp oligos on an agarose gel is going to be hard to see. If you have a mini-gel system available for polyacrylamide gels, you can always use a 20% TBE-PAGE gel to check your ligations. The gels run in 30 min, and you stain with EtBr, just like an agarose gel.

The only other advice I can give is try to keep your reaction volumes low, your oligo concentration high, AND your ligase concentration high. Promega sells a high concentration (HC) version of its T4 DNA ligase and I have used that for doing similar specialty ligations where I was volume limited.

Best of Luck.

#4 pgarcia

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Posted 14 October 2011 - 07:29 AM

Thanks very much,

The oligos are 5' XbaI---(70bp)---SpeI 3'.

The goal is get 6-12 copies in tanden of the oligos.

I use T4 DNA ligase from NewEngland Biolabs. I kill DNA ligase heating at 70ºC for 10 min. After that, run the reaction on agarose gel and only I get a smear, but I don't get any clear band. Maybe the problem is like allynspear said, the resolution on an agarose gel isn't very good. I'll try to do the same and run on polyacrylamide gels.
Thanks again and let me know if anybody has another idea.

#5 phage434

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Posted 14 October 2011 - 02:03 PM

With short fragments you probably would see a smear. the Invitrogen 20% TBE gels are easy to use and reliable for short fragments after EtBr staining, or you can make your own high percentage PAGE gels.

Note that you will not necessarily get tandem repeats with this set of enzymes. XbaI and SpeI have compatible ends, and you will get products with both orientations, along with some circular fragments. There's nothing much to do about the circular ones except to do the reaction at very high concentrations which will favor long linear products.
The orientation ambiguity could be controlled by adding XbaI and SpeI to your ligation mix to cut the end-to-end fragments that are formed.

#6 phage434

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Posted 14 October 2011 - 02:34 PM

Actually, to eliminate the circular fragments, you can either make a new pair of oligos with XbaI/SpeI ends and an internal restriction site or mess with phorphorylation. For the enzyme approach, mix your normal and RE containing fragments in the correct proportion. After ligation, purify and cut with this enzyme. This also provides a hook for inserting your fragment without blunt cloning. There will still be circular fragments, but there will also be linear fragments with the new RE ends.

You could do a similar thing with phosphorylation. Order normal (no 5' phosphate) oligos. Anneal them, then keep a portion (20%) while treating the remainder with PNK. Mix in the proportion of your choice to select the average fragment length, then ligate. You'll still get circular fragments with this approach, but you'll also get some linear fragments (with nicks). You'd need to ligate into a vector and repair in a cell, which is probably what you will do anyway.

#7 allynspear

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Posted 18 October 2011 - 07:10 AM

Gotta hand it to you, those are some very cool Ideas phage.

#8 pgarcia

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Posted 19 October 2011 - 05:50 AM

Thanks a lot, I'll try to do different ideas you tell me. Let you know about that.




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