Custom Primer - FAQs

I ordered two complimentary oligos in order to make an adapter. Do you have a protocol I can use to anneal them?

We recommend this protocol for annealing complimentary oligos to produce an adaptor. We also recommend that you PAGE-purify the oligos before you use them in adaptor annealing.

(1) Verify the oligo concentration by preparing duplicate dilutions and determining the A260. Concentration (nmole/mL) = A260 x nmole/OD x dilution factor total volume of oligo (mL)

(2) To prepare the adapter, add the following components together. (You will need to calculate volumes based on the concentrations of the oligonucleotides and total volume of the annealing reaction. You may scale the reaction to suit your needs.)
oligonucleotide 1, final concentration of 100 nmole/mL
oligonucleotide 2, final concentration of 100 nmole/mL
10X Annealing Buffer*, final concentration of 1X
DEPC-treated water to appropriate volume

(3) Bring the oligo solution to 65 degrees C by placing the tube in a 65 degrees C (or higher) waterbath. Maintain the oligo solution at 65 degrees C for 10 min. (It is critical to maintain the oligos at exactly 65 degrees C for the duration of this time.)

(4) Remove the solution from the water bath and allow it to cool slowly to room temperature (1-2 h).

(5) Store the adapter at -20 degrees C.

*10X Annealing Buffer:
Mix the following components for a 50 mL stock solution:
5 mL of 1 M Tris-HCl (pH 7.5) (final conc. = 100 mM)
10 mL of 5 M NaCl (final conc. = 1 M)
1 mL of 0.5 M EDTA (final conc. = 10 mM)
34 mL DEPC-treated water