Loading Concentration Calculator

Libraries

Enter the name of the library, starting concentration, starting volume, desired number of reads, and lanes the library will be pooled in. You can also copy the data from a spreadsheet.

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#	Name	Starting Concentration (nM)	Desired Reads (M)	Lane Number (1-16)
1

PhiX Control in all lanes % (between 0.1 and 5), starting concentration nM

Reagent Kit

Pick the flow cell and reagent kit.

Expected flow cell output: 150 M Reads, 15Gb

G4 Flow Cell	Kit Cycle Configuration	Output (Gb)	Reads (M)
F2 Flow Cell	F2 100 Cycle Kit	15	150
F2 Flow Cell	F2 200 Cycle Kit	30	150
F2 Flow Cell	F2 300 Cycle Kit	45	150

Loading Concentration

Set the desired loading concentration for every lane.

Lane Number	Flow Cell	Lane	Loading Concentration (pM)	Reads (M)

Pipetting Schedule

Make fresh 0.2 M NaOH by mixing 800 µL molecular-grade water with 200 µL 1 M NaOH and invert tube several times to mix. Use within 12 hours.

If the starting library concentration is higher than 4 nM, dilute the library in a microcentrifuge tube to 4 nM with TE. Vortex and then centrifuge briefly.

Library	Starting Concentration (nM)	Library Needed (µL)	Add TE (µL)	Total Volume(µL)	Final Concentration (nM)

Pool the libraries in separate new tubes. Vortex and then centrifuge briefly.

Pool for Lane	Library	Starting Concentration (nM)	Add Diluted Library(µL)	Total Volume (µL)

Denature the pools with 0.2 M NaOH in separate new tubes. Vortex and then centrifuge briefly.

Denature the libraries with 0.2 M NaOH. Vortex and then centrifuge briefly.

Pool for Lane	Libraries	Starting Concentration (nM)	Add Diluted Library(µL)	Add 0.2 M NaOH (µL)	Total Volume (µL)	Final Concentration (nM)

Incubate for 5 minutes at room temperature.

Neutralize the denatured librariespools by adding 200 mM Tris-HCl pH 7.0 to the same tubes, then vortex to mix and centrifuge briefly.

Pool for Lane	Starting Concentration (nM)	Volume Denatured Library (µL)	Add 200 mM Tris-HCl (µL)	Total Volume (µL)	Final Concentration (nM)

Dilute the neutralized librariespools to 200 pM by adding water to the same tubes (PhiX Control to 50 pM), then vortex to mix and centrifuge briefly.

Pool for Lane	Starting Concentration (nM)	Volume Neutralized Library (µL)	Add Water (µL)	Total Volume (µL)	Final Concentration (pM)

Place on ice until ready to proceed.

Dilute the 200 pM librariespools Combine the 200 pM librariespools and the 50 pM PhiX Control and dilute to the loading concentration in 300 µL per lane with 2xSample Loading Buffer (2xSLB) in a new tube. Vortex and then centrifuge briefly.

Pool for Lane	Volume LibraryPool (µL)	PhiX (µL)	Volume Water (µL)	Volume 2xSLB (µL)	Total Volume (µL)	Final Concentration (pM)

Keep on ice until ready to use, no longer than 2 hours.

When loading the sample cartridge, pipette 260 µL of the pool at loading concentration into the appropriate well for each lane.

Expected Number of Reads

Expected number of reads (in million of reads) for each library in each lane.

Pool for Lane
Flow Cell
Lane