oligo annealing - (Oct/30/2006 )

Hi,

I have to anneal oligoa 16 and 12 bp long and phosphorlate it before ligatation to the target sequence.
Can anybody help me with the annealing buffer ingredients and the procedure?

Since I need to phosphorlate the annealed oligo before ligation I should add kinase with kinase buffer.So how do I purify the annealed oligo from the annealing buffer?

Please reply

10 X annealing buffer:

100 mM Tris HCl, pH 8
500 mM NaCl
10 mM EDTA

Annealing Protocol:

Place in a heat block at exactly 95 degrees C for 10 min.
Spin Briefly
Place back in heat block and turn OFF
Allow block to reach 25 degrees C.

My annealing mixture:

Oligo 1 10 ul (30 ug)
Oligo 2 10 ul (30 ug)
10 X annealing buffer 3 ul
H20 7 ul
total = 30 ul

This protocol was not for phosphorylation, so I don't know if the ug amounts would be right for your purpose, but these amounts worked for me (I was making an adaptor for ligation into my vector).
After annealing, check the components of your kinase buffer, if it is similar to the annealing buffer you can use a small amount of the annealing mixture in the kinase reaction.
When I purified my annealed oligos I used Phenol/chl extraction.

Sorry Zona, I just go the quick and dirty route.
Make up the oligos in the kinase buffer heat to 95 in a thermocycler for 1 minute and let it cool however it fast it likes. (Remember, the oligos are only 12 and 16 bp long.)
When it's cooled down to RT, add kinase and run the reaction as normal.

As for purifying the annealed, phosphorylated linker, you could try gel filtration (try a peptide column), or maybe ammonium acetate/EtOH pptn (but be prepared for some losses). However, I'd just add it at the required volume straight into the ligation reaction. That, of course requires you to resuspend the original oligos at such a concentration that you can add a small volume to the ligation reaction.
Works for me.

Thanks guys....Im going to try both methods..I will write as soon it works.