Urine Conditioning Buffer (UCB) Treatment

Complete the following steps to perform UCB treatment, the first step to extract DNA before library preparation.

Always cap and seal tubes before centrifuge steps.

• ZR BashingBead Lysis Tube
• Zymo-Spin III-Filter
• Zymo-Spin IC-S Column
• 2.0 ml collection tubes
• 1.5 ml microcentrifuge tube
• 2.0 ml microcentrifuge tube
• UCB (Urine Conditioning Buffer)
• Clearing Beads
1. Remove samples and external control set from storage, and prepare as follows.
a. Incubate at room temperature for 30 minutes.
b. Vortex and centrifuge briefly to mix.
2. Remove T7 Phage stock from storage, and prepare as follows.
a. Generate a Working stock concentration of 1.09E+10 copies/ml by diluting the purchased T7 Phage stock with water, if necessary.
b. Vortex and centrifuge briefly to mix. Place on ice.
c. (Optional) Dispense the T7 Phage Working stock into aliquots of 50 µl (or other volumes) and frozen to avoid multiple freeze thaws of the Working stock, and simplify usage in future experiments.
3. For each sample to be extracted, including the external control samples, prepare the following labeled tube sets.

Tube

Quantity

ZR BashingBead Lysis Tube

1

Zymo-Spin III-Filter

1

Zymo-Spin IC-S Column

1

2.0 ml microcentrifuge tube

2

1.5 ml microcentrifuge tube

1

2.0 ml collection tube

5
1. Add 1.8 ml urine sample to a new 2.0 ml microcentrifuge tube.
2. Add 126 µl UCB to each microcentrifuge tube. Vortex to mix.
3. Add 10 µl Clearing Beads to each microcentrifuge tube. Vortex to mix.
4. Centrifuge the microcentrifuge tubes at 3000 × g for 15 minutes.
5. Without disturbing the bead pellet, use a single channel pipette to remove and discard 1500 µl of supernatant.
6. Vortex or pipette thoroughly to resuspend the pellet in the residual solution.