3. Prepare a fresh dilution of 0.2 N NaOH (combine 980μL of
nuclease-free water and 20μL of 10 M NaOH in a
LoBind tube).
4. Prepare PhiX control (see Note 27) by adding 2μL of 10 nM
PhiX library with 3μL of 10 mM Tris–Cl, pH 8.5 with 0.1%
Tween 20 in a LoBind tube. Mix well. Then, add 5μL of 0.2 N
NaOH, briefly vortex, and then centrifuge at 280 Â g for
1 min. Incubate for 5 min at room temperature and then add
990μL of prechilled HT1 buffer. This is 20 pM denatured
PhiX, and for v2 reagents, 12.5 pM is required: combine
375μL of 20 pM denatured PhiX with 225μL of prechilled
HT1 (see Notes 28 and 29).
5. Denature 5μL of the 2 nM library pool by combining with 5μL
of 0.2 N NaOH, briefly vortex, and then centrifuge at 280 Â g
for 1 min. Incubate for 5 min at room temperature and then
add 990μL of prechilled HT1 buffer. This is a 10 pM denatured
library pool; dilute to 8 pM by combining 800μL of 10 pM
denatured library pool with 190μL of prechilled HT1 buffer
and 10μL of 12.5 pM denatured PhiX. Mix well.
6. Add 600–1000μL of the denatured and diluted library pool +
PhiX mix to the designated reservoir (labeled as LOAD SAMPLES) on the thawed reagent cartridge.
7. From the MiSeq Welcome Screen, select ‘Sequence’ and then
follow the run setup steps.
8. Remove flow cell from storage buffer using plastic forceps.
Rinse the flow cell under running distilled water to remove
excess salts, dry thoroughly using a lint-free tissue, and clean
the flow cell glass with an alcohol wipe to ensure the glass is free
for streaks, fingerprints, or fibers. Avoid touching the flow cell
port gasket (black rubber area at the base of the flow cell).
Allow to dry and load onto MiSeq following the screen
prompts.
9. Load fresh PR2 bottle and ensure the waste bottle is empty,
following the screen prompts.
10. Load reagent cartridge into the chiller, following the screen
prompts.
11. Change the sample sheet name to match bar code on reagent
cartridge, review the run parameters, and prerun check results.
12. Select Start Run (see Note 30).
13. When run is complete, perform postrun washes, as per standard MiSeq maintenance.
14. Export fastq files for analysis using software of choice; we favor
CLC Genomics Workbench from Qiagen.
476
Amanda Claire Brown
nuclease-free water and 20μL of 10 M NaOH in a
LoBind tube).
4. Prepare PhiX control (see Note 27) by adding 2μL of 10 nM
PhiX library with 3μL of 10 mM Tris–Cl, pH 8.5 with 0.1%
Tween 20 in a LoBind tube. Mix well. Then, add 5μL of 0.2 N
NaOH, briefly vortex, and then centrifuge at 280 Â g for
1 min. Incubate for 5 min at room temperature and then add
990μL of prechilled HT1 buffer. This is 20 pM denatured
PhiX, and for v2 reagents, 12.5 pM is required: combine
375μL of 20 pM denatured PhiX with 225μL of prechilled
HT1 (see Notes 28 and 29).
5. Denature 5μL of the 2 nM library pool by combining with 5μL
of 0.2 N NaOH, briefly vortex, and then centrifuge at 280 Â g
for 1 min. Incubate for 5 min at room temperature and then
add 990μL of prechilled HT1 buffer. This is a 10 pM denatured
library pool; dilute to 8 pM by combining 800μL of 10 pM
denatured library pool with 190μL of prechilled HT1 buffer
and 10μL of 12.5 pM denatured PhiX. Mix well.
6. Add 600–1000μL of the denatured and diluted library pool +
PhiX mix to the designated reservoir (labeled as LOAD SAMPLES) on the thawed reagent cartridge.
7. From the MiSeq Welcome Screen, select ‘Sequence’ and then
follow the run setup steps.
8. Remove flow cell from storage buffer using plastic forceps.
Rinse the flow cell under running distilled water to remove
excess salts, dry thoroughly using a lint-free tissue, and clean
the flow cell glass with an alcohol wipe to ensure the glass is free
for streaks, fingerprints, or fibers. Avoid touching the flow cell
port gasket (black rubber area at the base of the flow cell).
Allow to dry and load onto MiSeq following the screen
prompts.
9. Load fresh PR2 bottle and ensure the waste bottle is empty,
following the screen prompts.
10. Load reagent cartridge into the chiller, following the screen
prompts.
11. Change the sample sheet name to match bar code on reagent
cartridge, review the run parameters, and prerun check results.
12. Select Start Run (see Note 30).
13. When run is complete, perform postrun washes, as per standard MiSeq maintenance.
14. Export fastq files for analysis using software of choice; we favor
CLC Genomics Workbench from Qiagen.
476
Amanda Claire Brown