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 (1.5 ml)
• Nuclease-free water (1.5 ml)

These volumes produce 3 ml 0.5X PWB1 per sample.

2. Centrifuge the PIP tubes for 1 minute.
3. Without disturbing the pellet, remove and discard 300 µl supernatant.

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

4. Wash PIPs as follows.
a. Add 1 ml 0.5X PWB1.
b. Vortex for 5 seconds.
c. Centrifuge for 30 seconds and return to the 1.5 ml tube holder.
d. Without disturbing the pellet, remove and discard 800 μl supernatant.
5. Repeat steps 4 a–d to wash PIPs a second time. Use 800 μl 0.5X PWB1.
6. Repeat steps 4 a–d to wash PIPs a third time. Use 800 μl 0.5X PWB1.
7. Weigh and record the total mass of each tube with PIPs to convert it to volume. Assume density is 1g/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

 

 

 

 
8. Centrifuge for 30 seconds.
9. 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 volume is 250 µl.

10. Store samples on ice and proceed immediately to cDNA amplification.