Clean Up PCR Product

•

IPB (Illumina Purification Beads)

•

RSB (Resuspension Buffer)

•

EtOH (absolute ethanol)

•

IPB (Illumina Purification Beads)

–

Vortex frequently to make sure of even distribution.

–

Aspirate and dispense slowly due to the viscosity of the solution.

Prepare the following consumables:

•

IPB—Vortex to mix.

•

RSB—Thaw on ice. Vortex and invert to mix.

•

80% EtOH—Prepare 400 µl 80% EtOH from absolute EtOH for each well of PCR product.

1.

Vortex and add 15 µl of IPB to each 25 µl RT-PCR reaction.

2.

Shake at 2200 rpm for 1 minute.

3.

Incubate at room temp for 5 minutes.

4.

Centrifuge at 280 x g for 3 seconds. Place on a magnetic stand and wait until the liquid is clear.

5.

Remove and discard all supernatant.

6.

Wash beads as follows:

a.

Add 175 µl 80% EtOH to each well.

b.

Wait for 30 seconds.

c.

Remove all supernatant and discard.

7.

Wash beads a second time.

8.

Dry the beads at room temperature for 2 min.

9.

Add 23 µl RSB to each well, and remove from the magnetic stand.

10.

Shake at 2200 rpm for 1 minute.

11.

Incubate at room temperature for 2 minutes.

12.

Centrifuge at 280 x g for 3 seconds.

13.

Place on a magnetic plate stand and wait until liquid is clear.

14.

Transfer 20 µl of supernatant from each well to the new PCR plate labeled TAG1.