Prepare RMR
1
Invert to mix.
2
Centrifuge at 1000 rpm for 1 minute.
Do not vortex.
3
Set aside on ice.
Prepare ICB for Read 2 (Non-Index or Single-Index)
1
Remove 2 tubes of LFN from -25°C to -15°C storage and thaw in a beaker filled with room temperature
deionized water for about 20 minutes.
2
When thawed, add the contents of the 2 tubes of LFN to the bottle containing 47 ml ICB.
3
Rinse each LFN tube with ICB to make sure that all LFN is transferred.
4
Add 1.1 ml EDP to the ICB and LFN solution.
5
Return any unused portion of EDP to -25°C to -15°C storage.
6
Cap the bottle containing EDP, ICB, and LFN and invert to mix.
7
Set aside on ice.
Prepare ICB for Read 2 (Dual-Index)
Use the following instructions to calculate and prepare the volume of ICB required for Read 2 of a dual-
indexed run.
1
Measure the following volumes of EDP, ICB, and LFN for every 10 cycles of sequencing to be performed
in Read 2.
Reagent
Volume per 10 Cycles
Storage
ICB
4.57 ml
2°C to 8°C
LFN
0.6 ml
-25°C to -15°C
EDP
0.11 ml
-25°C to -15°C
For more information on calculating volumes, see
2
Transfer the measured volume of ICB to an empty 250 ml bottle.
3
Add the measured volume of LFN to the new bottle of ICB.
4
Rinse each tube of LFN with ICB to make sure that all LFN is transferred.
5
Add the measured volume of EDP to the ICB and LFN solution.
6
Rinse the tube of EDP with the ICB and LFN solution to make sure that all EDP is transferred.
7
Cap the bottle containing EDP, ICB, and LFN and invert to mix.
8
Set aside on ice.
Load Paired-End Reagents
1
Make sure that the paired-end rack is not in use on the opposite flow cell for Read 2 resynthesis, Index 1
(i7) Read preparation, or Index 2 (i5) Read preparation.
2
Raise the sippers for the paired-end reagent rack as follows.
Document # 15011190 v03
For Research Use Only. Not for use in diagnostic procedures.
41
HiSeq 2000 System Guide