Prepare and Add Custom Primers

Prepare custom primers using Hybridization Buffer (HT1) and then add them to the custom primer (CP) wells on the MiSeq i100 Plus dry cartridge. HT1 is not provided but can be purchased separately, refer to User-Supplied Consumables & Equipment.

Prepare Custom Primers

1.

If frozen, thaw each custom primer to be used.

2.

If using custom or third-party libraries only, prepare them as follows:

•

Use HT1 to dilute the custom read primer to yield a total volume of 500 µl, with each custom read primer at 0.3 µM final concentration.

•

Use HT1 to dilute the custom index primer or index primer mix to yield a total volume of 500 µl, with each custom index primer at 0.6 µM final concentration.

3.

If using custom or third-party libraries with PhiX or Illumina libraries, prepare custom read primers, or custom index primers as follows:

•

Add each custom read primer mix to 500 μl VP21 or HP21 for a 0.3 μM final concentration.

•

Add each custom index primer mix to 500 μl VP14 or BP14 for a 0.6 μM final concentration.

Add Custom Primers to the Dry Cartridge

Refer to Dry Cartridge for well locations.

1.

Using a clean pipette tip, pierce the foil seal covering the appropriate CP well on the dry cartridge.

2.

Add 500 uL custom primer to the appropriate well.

Dispense the liquid slowly to avoid spillage, bubbles, and cross-contamination.

•

CP1—Reagent Port for loading Custom Read 1 Primers.

•

CP2—Reagent Port for loading Custom Read 2 Primers.

•

CP3—Reagent Port for loading Custom Index Primers.