annealing oligos - (Jul/29/2014 )

Hallo all,

I have some oligos to anneal to clone into a plasmid.

The problem is however that one of the people that also uses these dissolved them in the wrong buffer.

We normally use an annealing buffer of 1mM EDTA, 50mM NaCl and 10mM Tris however the buffer they used to dissolved the oligos contained the wrong EDTA concentration: 25 times the amount : so 25mM rather than 1mM.

Anyone an idea if this would be a problem if I tried to make the oligos to anneal? 

I could add some more NaCl and Tris to dilute the EDTA , however I would have to dilute the oligos a lot than and I think they would not anneal anymore if I dilute it too a concentration that is rather low.

Now I have a 100micromolar concentration of oligos, diluting this 25 times seems a bit too much since I would end up with just 4 micro molar of the oligos.

Any insight?

I suspect that the EDTA is most likely to interfere with the ligation step.  You could try a buffer exchange by dialysis, but it is probably cheaper and easier to just order new oligos.

The EDTA will not inhibit the annealing, but as Bob1 says, downstream use of the oligos will likely be a problem due to the high EDTA concentration.

Ok thanks for the information.

I will dilute them 10 times for the ligation, so I will end up with 2.5 mM EDTA, still more than twice what is needed (1mM) but perhaps this will not be such a big problem.

I will try it and if needed order new oligos.