Prepare PhiX for a Troubleshooting Run
Sequencing System: NovaSeq X
Use the following procedure to denature and dilute a PhiX library for use as a PhiX-only sequencing run. Performing a PhiX-only run is helpful in confirming instrument performance or for troubleshooting purposes. A PhiX-only run requires 100% PhiX library at recommended volumes and loading concentration.
This protocol requires the following consumables and equipment.
Consumables
|
Consumables |
Supplier |
Purpose |
|---|---|---|
|
1 N NaOH |
General lab supplier |
Diluting to 0.2 N for denaturing libraries. |
|
Microcentrifuge tube, 1.5 ml |
VWR, catalog # 20170-038, or equivalent |
Combining volumes. |
|
Pipette tips, 20 μl |
General lab supplier |
Pipetting reagents and libraries. |
|
Pipette tips, 200 μl |
General lab supplier |
Pipetting reagents and libraries. |
|
Pipette tips, 1000 μl |
General lab supplier |
Pipetting reagents and libraries. |
|
Pre-load buffer |
Illumina, provided in the NovaSeq X Series reagent kit |
Neutralizing denatured libraries. |
|
Resuspension Buffer (RSB) |
Illumina, provided in the library prep kit contents |
Diluting libraries to the required loading concentration. |
|
Water, laboratory-grade |
General lab supplier |
Diluting NaOH for denaturing libraries. |
|
PhiX Control v3 |
Illumina, catalog # FC-110-3001 |
PhiX-only run for troubleshooting. |
Equipment
|
Equipment |
Supplier |
|---|---|
|
Microcentrifuge |
General lab supplier |
|
Vortexer |
General lab supplier |
Prepare NaOH
Prepare a fresh dilution of 0.2 N NaOH to denature PhiX for sequencing. Extra volume is prepared to prevent small pipetting errors from affecting the final NaOH concentration.
| 1. | Combine the volumes appropriate for your flow cell type in a microcentrifuge tube. Multiply each volume by the number of flow cells. |
|
Flow Cell Type |
Laboratory-grade water (µl) |
Stock 1 N NaOH (µl) |
Total (µl) |
|---|---|---|---|
|
25B |
120 |
30 |
150 |
|
10B or 5B |
80 |
20 |
100 |
|
1.5B |
20 |
5 |
25 |
| 2. | Vortex to mix, and then centrifuge briefly. |
Dilute PhiX
| 1. | Quantify PhiX |
| 2. | Dilute PhiX to 2 nM using RSB. |
| 3. | In a new 1.5 ml microcentrifuge tube, dilute PhiX to a 140 pM final loading concentration using RSB. |
|
Flow Cell Type |
2 nM PhiX (µl) |
RSB (µl) |
Total |
|---|---|---|---|
|
25B |
19.6 |
36.4 |
56 |
|
10B, 5B, or 1.5B |
11.9 |
22.1 |
34 |
| 4. | Vortex to mix, and then centrifuge briefly. |
Denature PhiX
| 1. | Add the appropriate volume of 0.2 N NaOH to the nondenatured PhiX tube. |
|
Flow Cell Type |
0.2 N NaOH (µl) |
|---|---|
|
25B |
14 |
|
10B, 5B, or 1.5B |
8.5 |
| 2. | Vortex to mix, and then centrifuge briefly. |
| 3. | Incubate at room temperature for 5 minutes to denature. |
| 4. | Add pre-load buffer to neutralize. |
|
Flow Cell Type |
Pre-Load Buffer (µl) |
|---|---|
|
25B |
210 |
|
10B, 5B, or 1.5B |
127.5 |
| 5. | Vortex to mix, and then centrifuge briefly. |
| 6. | Store libraries on ice until transferred to the library tube strip. |
After denaturing and diluting the libraries, you are ready to load the lyo insert and library tube strip into the reagent cartridge in preparation for sequencing. Refer to the sequencing system product documentation for more information.
Revision History - Protocol for PhiX Run
|
Document |
Date |
Description of Change |
|---|---|---|
|
Document # 200056655 v01 |
May 2026 |
Added information for NovaSeq X Series 5B Flow Cell. Added centrifuge step after diluting libraries. Updated vortex and centrifuge steps for consistency. |
|
Document # 200056655 v00.1 |
May 2025 |
Corrected typo. |
|
Document # 200056655 v00 |
June 2024 |
Initial release. |