Annealing two Primers - PCR (Jan/14/2007 )

Dear all,
I am still new here. I am Hosea from the Netherlands. I am having a problem. Basically I wanted to order ds oligo but it is rather expensive therefor I ordered 2 primers which are complementary to each other. Each primer has 45 nucleotides. How to anneal both of them toegther? Are there any spesific condition or protocols? Thanks for your help!


Groetjes

U could check invitrogen or www.idtdna.com for protocols.

We follow invitrogen's protocol for annealing oligos.

recipe for 10X Oligo Annealing Buffer

100 mM Tris-HCl, pH 8.0
10 mM EDTA, pH 8.0
1 M NaCl

Dear Scolix,
Thank you for your reply, I would try to do that. If I see from Invitrogen protocol, we need to make sure that:
“Top strand” oligo: Make sure that this oligo contains the sequence, CACC, at the 5' end.
“Bottom strand” oligo: Make sure that this oligo contains the sequence, AAAA, at the 5' end and is complementary to the top strand oligo.

Have you had any experience if the two single strands do not have the sequences? Any affects?

Thanks.

Hi Hosea,

you can also try the annealing this, way, I always do it for my gelshifts, works fine:


10x annealing buffer:
200 mM Tris pH 7,6
100mM MgCl
500 mM NaCl

Reaction mixture:
- 50 µg Oligo A
- 50 µg Oligo B
- 26µg 10x Annealing Puffer
- 134 µl TE
Heat reaction mixture to 70°C for 5 min, let it cool down slowly to room temperature (over night, in the heater, power turned down). Then you can store it in -20°C.

I just made a solution of the two primers in an eppendorf-cup (a safe lock).
Then I took a large beaker with water, on a burner and wait till it boils, then turn off the burner and add the primer-mix (keep it in the eppendorf-cup, so just drop the cup in) to the water when it just stopped boiling. Then leave it overnight.
The slow cooling works great for annealing primers to each other.

Dont worry abt that. Invitrogen'manual is for annealing oligos for hairpin loops (shRNA). thats y the specific sequence.

For annealing oligos, just take equal amounts and go ahead with the annealing. I have annealed other oligos similar way.

i anneal oligos now in PCR apparatus as in thermo blocks, evaporation drive enhancment of salt concentration.