Wash PIPs for cDNA Amplification

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• PWB1 (Wash Buffer)
• Nuclease-free water (customer provided)
1. Combine the following volumes to prepare 0.5X PWB1. Multiply each volume by the number of samples.
• PWB1 (6 ml)
• Nuclease-free water (6 ml)

These volumes produce 12 ml 0.5X PWB1 per sample.

2. Centrifuge the PIP tubes in a swinging bucket rotor at 750 × g (70–80% maximum braking speed) for 2 minutes.
3. Without disturbing the pellet, remove and discard supernatant to the 1 ml mark on the tube.

If an aspirator is used for supernatant removal, change tips with each aspiration.

4. Wash PIPs as follows.
a. Add 4 ml 0.5X PWB1.
b. Vortex for 5 seconds.
c. Centrifuge for 2 minutes.
d. Without disturbing the pellet, remove and discard supernatant to the 1 ml mark on the tube.
5. Repeat steps 4 a–d to wash PIPs a second time. Use 4 ml 0.5X PWB1.
6. Repeat steps 4 a–d to wash PIPs a third time. Use 4 ml 0.5X PWB1.
7. Store samples on ice and proceed immediately to cDNA amplification.