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How should I anneal longer hairpin oligos?

This is a protocol that has been used to clone >10,000 hairpin siRNA templates into a plasmid vector for siRNA expression at GNF in San Diego. They use desalted oligos and find by sequencing that over half the clones are "perfect" and note that using purified oligos will improve the "hit rate" but that for high throughput work it is not necessary. They believe that using a good anneal/clone protocol is crucial to their high success rate. They also include a kinasing step in this procedure if needed. If you do not need to kinase your oligos, you can resuspend them to equimolar concentrations and skip to step 5.

1. Bring sense and antisense oligos to 10 pmol/ul by adding 100 ul r.o.-quality water per well (oligos started as separate plates, normalized to 1 nmol per well, and were sent to us lyophilized).

2. Mix 2 ul of sense and antisense together in a 96-well thin-walled PCR plate.

3. Bring to 20 ul final volume with 5x Polynucleotide Kinase Buffer (1x final), which, at 1x final consists of 70 mM TrisHCl, pH 7.6, 10 mM MgCl2, 100 mM KCl, 1 mM BME. Since I kinased the oligos, which shouldn't be necessary but which I did just to ensure that the experiment would work, the PNK mix also contained 20 mM ATP (not dATP!) and 10 units T4 polynucleotide kinase (oligo concentration was now 1 pmol/ul).

4. Incubated kinase reaction 10 min at 37°C and then heat killed PNK by incubating 10 minutes at 65°C.

5. Annealed using a modified touchdown protocol on a PTC-100 cycler as follows: 95°C for 30s, 60°C for 10 min, then cool to 20°C at 1 degree every 15s.

6. Aliquot 2 ul of annealed, phosphorylated oligos to a 96 well plate.

7. Add 38 ul of water, bringing the final oligo concentration fo 0.05 pmol/ul.

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