Prepare Dry Cartridge

MiSeq i100 Series consumables are shipped and stored at room temperature. Thawing is not required. Before loading the libraries into the dry cartridge, dilute libraries and optionally spike inPhiX. Libraries are denatured automatically onboard the instrument.

Always perform a quality control analysis and optimize the loading concentration for your library.

Dilute Libraries

Use scissors to cut open the wet cartridge foil packaging to retrieve the Resuspension Buffer (RSB) and Library Denaturation Buffer (KLD) tubes. Set the tubes aside.

Keep the wet cartridge in the foil packaging until it is ready to be loaded. The wet cartridge must be used within 4 hours of opening the foil packaging.

Dilute libraries to 10x loading concentration to a total volume of 30 µl using RSB.

Example: For a final loading concentration of 100 pM, dilute to 1 nM.

Vortex at the highest setting for 3 seconds, and then centrifuge briefly.

[Optional] Spike in PhiX as follows.

For intended PhiX spike-in ≥10%, dilute PhiX to 10x library loading concentration with RSB and combine with 10x library solution to a total volume of 30 µl. Use appropriate volumes of PhiX and library to produce desired PhiX spike-in percentage.

Example: Add 3 µl 10x PhiX solution to 27 µl 10x concentration libraries to obtain 30 µl 10x library mixture with 10% PhiX spike-in.

For intended PhiX spike-in <10%, dilute PhiX to 6x library loading concentration with RSB and combine with 10x library solution to desired spike-in percentage.

Example: For a final loading concentration of 100 pM, dilute PhiX to 0.6 nM with RSB and add 1 µl PhiX mixture to 29 µl 10x loading concentration library mixture.

The volumes produce approximately a 2% PhiX spike-in. The percentage varies depending on the library quality and quantity.

In a new 1.5 ml microcentrifuge tube, combine the following volumes to dilute libraries to the final loading concentration:

•

10x loading concentration library (30 µl)

•

KLD (270 µl)

Vortex at the highest setting for 3 seconds, and then centrifuge briefly.

Store mixture on ice until ready for use.

The diluted library solution is stable up to 6 hours when stored on ice or at 4ºC.

Load Libraries

Put on a new pair of powder-free gloves to avoid contamination.

Use scissors to cut open the dry cartridge foil packaging.

Use the dry cartridge within 4 hours of opening the foil packaging.

Remove the dry cartridge from the package.

Grasp the dry cartridge by the sides to avoid touching the flow cell.

Discard the foil package in accordance with the applicable local standards.

Using a clean pipette tip, pierce the foil seal covering the reagent well labeled Library.

Pipette 250 µl diluted library solution into the Library well in the dry cartridge.

[Optional] Pipette custom primer into the appropriate port on the dry cartridge. Refer to Custom Primers.