How to anneal more than 2 oligos???? - Protocol wanted that allows the annealing of several oligos (Aug/03/2010 )
I am desperate because of the oligo annealing and hope that someone of you can help me!
I am trying to anneal more than 2 oligos (up to 4 oligos each with a length of about 30 bp) at the same time to get a longer DNA sequence. I used several times our standard protocol that works very good for the annealing of 2 (waterbath), but it does not work for my current purpose. Does anybody has an idea how I can manage this?
Thank you very much in advance!
I think there is a bit of confusion regarding what you are trying to do with your oligos. Generally, when you anneal 2 oligos what you are doing is taking a single-stranded sense and a complimentary single-stranded anti-sense piece of DNA and annealing them to create a double stranded product. The length of the DNA strand is not longer. Rather, it sounds like you are wanting to ligate your oligos end-to-end to create a longer DNA product. Is this correct?? If so, can you tell us more about the DNA you are working with? Is it single stranded? Are there compatible ends? I've never tried or even heard of trying to ligate single stranded, blunt end DNA but perhaps someone else here can help you if you clarify what it is you are trying to do.
There are at least two ways to anneal more than two oligos.
But first, it is important that the oligos do overlap (the mor the better - half would be perfect ) :
5' __________ _____________ _____________ 3'
3' _____ ___________ _____________ __________ 5'
The first method uses PCR: You put together all oligos, which function as templates and primers as well here, and set up a usual PCR reaction where you only use ten cycles.
Another method needs phosphorylation of your oligos to get them linked together later when you do ligation.
You can phosphorylate all oligos in one tube and the anneal them using 10x "annealing buffer" (100 mM Tris-HCL pH8.0; 10 mM EDTA pH8.0; 1 M NaCl) and filling up the volume with water.
Then you heat up your sample to 70°C for 5 minutes and let it cool down slowly to RT which may take two to three hours.
Both methods work but I prefer the PCR one.