Wash and Prepare PIPs

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• Washing Buffer (PWB1)
• Nuclease-free water
• 0.2 ml 8-tube strip and caps
1. Prepare the PIP pellet as follows.
a. Centrifuge briefly to collect contents.
b. Place on ice.
1. Combine the following volumes to prepare 0.5X PWB1. Multiply each volume by the number of samples.
• PWB1 (300 µl)
• Nuclease-free water (300 µl)

These volumes produce 600 μl 0.5X PWB1 per sample.

2. Wash the PIPs as follows.
a. Add 120 μl 0.5X PWB1.
b. Vortex for 5 seconds.
c. Centrifuge for 5 seconds.
d. Place into the guide rack, and then tap the rack against the bench three times.
e. Let sit until the PIPs settle below the guide wire (~1 minute).
f. If PIPs remain above the guide wire, tap the strip again and wait (~1 minute).
g. Without disturbing the pellet, remove and discard 150 μl supernatant.
3. Repeat wash a second time. Use 150 μl 0.5X PWB1.
4. Repeat wash a third time. Use 150 μl of 0.5X PWB1.
5. Repeat wash a fourth time. Use 150 μl of 0.5X PWB1.
6. Centrifuge for 5 seconds.
7. Let sit until the PIPs settle below the guide wire (~1 minute).
8. Remove and discard supernatant to the guide wire.
9. Place samples on ice, and immediately proceed to sgRNA amplification.