Wash and Prepare PIPs

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• Washing Buffer (PWB1)
• Nuclease-free water
• 1.5 ml PCR Clean centrifuge tube
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 (1.5 ml)
• Nuclease-free water (1.5 ml)

These volumes produce 3 ml 0.5X PWB1 per sample.

2. Wash the PIPs as follows.
a. Add 1 ml 0.5X PWB1.
b. Vortex for 5 seconds.
c. Centrifuge for ~30 seconds.
d. Place the tubes in the 1.5 ml side of the combination tube stand.
e. Without disturbing the pellet, remove and discard 800 μl supernatant.
3. Repeat wash a second time. Use 800 μl 0.5X PWB1.
4. Repeat wash a third time. Use 800 μl of 0.5X PWB1.
5. Weigh and record the mass of an empty 1.5 ml tube.

Make sure to weigh the same kind of tube that is being used to store the washed PIPs.

6. Weigh and record the total mass of each tube with PIPs to convert it to volume. Assume that density is 1 g/ml. Refer to the following example to calculate the aspiration volume necessary to normalize each mixture volume to 250 µl. One empty tube mass can be representative of all empty tubes.

Measurements

How to calculate

Sample 1

Sample 2

Sample 3

Sample 4

Total mass (mg)

Empty tube mass+PIPs

 

 

 

 

Empty tube mass (mg)

1.5 ml empty tube mass

 

 

 

 

PIP solution volume (μl)

Total mass‑empty tube mass=PIP mass/1

(equation converts PIP mass to PIP solution volume in µl)

 

 

 

 

Supernatant to remove (μl)

PIP solution volume-250 μl

 

 

 

 
7. Centrifuge for 30 seconds.
8. To remove the calculated volume of supernatant, aspirate twice as follows.
a. Tip the tube, and then aspirate and discard half of the calculated volume.
b. Centrifuge for 30 seconds.
c. Tip the tube, and then aspirate and discard the remaining supernatant.

The total remaining volume is 250 µl.

9. Place samples on ice, and immediately proceed to sgRNA amplification.