u
#5 SB2
u
#6 SB2
u
#7 CMR
u
#8 SB3
3
Add the following volume of each reagent to the appropriate 250 ml bottle to result in a second set of
reagents. Prepare CMR last and then replace your gloves.
Number
Reagent Name
Volume
1
ICB
47 ml
3
SRE
107 ml
4
SB1
100 ml
5
SB2 (for position #5)
62 ml
6
SB2 (for position #6)
62 ml
7
CMR
107 ml
8
SB3
110 ml
4
Use both sets of reagents for 2 concurrent runs or store 1 set at the following temperatures:
u
Store CMR, SRE, and EDP at -25°C to -15°C.
u
Store ICB, SB1, SB2, and SB3 at 2°C to 8°C.
Prepare ICB
1
Add the contents of 2 tubes of LFN to 1 bottle of ICB.
2
Rinse each tube of LFN with ICB to make sure that all LFN is transferred.
3
Add 1.1 ml EDP to the ICB and LFN solution.
4
Return the unused portion of EDP to -25°C to -15°C storage.
5
Cap the bottle containing EDP, ICB, and LFN and invert to mix.
6
Set aside on ice.
Primer Rehybridization
A rehybridization run repeats the sequencing primer hybridization step. If run metrics indicate low cluster
numbers, low cluster intensities, or other concerns, perform primer rehybridization to rescue the flow cell.
Primer rehybridization does not damage clusters.
HiSeq v4 Flow Cell
All rehyb steps are performed on the HiSeq 2000. The kit includes primers for Read 1, Index 1 Read, Index 2
Read for single-read flow cells, and Read 2.
Rehyb Kit Name
Workflow Instructions
HiSeq Multi-Primer Rehyb Kit v4
Catalog # GD-403-4001
HiSeq High Output Primer Rehybridization Guide (part
# 15050105)
Document # 15011190 v03
For Research Use Only. Not for use in diagnostic procedures.
54
HiSeq 2000 System Guide