self-ligation Anneled oligos
Posted 13 October 2011 - 09:01 AM
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,
Posted 13 October 2011 - 03:50 PM
Posted 14 October 2011 - 06:17 AM
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.
Posted 14 October 2011 - 07:29 AM
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.
Posted 14 October 2011 - 02:03 PM
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.
Posted 14 October 2011 - 02:34 PM
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.
Posted 18 October 2011 - 07:10 AM
Posted 19 October 2011 - 05:50 AM