Protocol #06
MiniSeq denature and dilute guide
Standard manual normalization
Prep:
Prepare reagents
 00 Cartridge needs to be thawed a day prior to sequencing. Store at 4 °C.
 01 Prepare a fresh 0.1 N dilution of NaOH from the 10 N stock solution.
Perform a two-step, ten-fold dilution with water. The stock solution is viscous. Ensure
proper mixing. Quick spin.
 02 Thaw the HT1 Hybridization Buffer. Quick vortex and spin. Keep it on ice before use.
 03 Thaw the PhiX sequencing control. Quick vortex and spin. Keep it on ice before use.
 04 Dillute sequencing primers to 50 µM with water from their 100 µM stock.
 05 Denature the sequencing primers by heating them to 95 °C for three minutes in a
cycler with a heated lid to 105 °C.
 06 Immediately place the primers into ice. Do not allow them to cool slowly.
Library
 07 Dilute the pooled and quantified library to 1 nM using the RSB buffer.
 08 Combine 5 µL of the 1nM library and 5 µL of the 0.1 N NaOH.
 09 Quick vortex and quick spin.
 10 Incubate at room temperature for 5 minutes.
 11 Add 5 µL of 200 µM Tris-HCl, pH 7.0
 12 Quick vortex and quick spin.
 13 Add 985 µL of chilled Hybridization Buffer to the denatured library. Vortex before.
The total volume is 1 mL at 5 pM concentration.
 14 Quick vortex and quick spin.
 15 Transfer 140 µL of the diluted library to a new tube.
 16 Add 360 µL of chilled Hybridization Buffer. Vortex before use.
The total volume is 500 µL at 1.4 pM concentration.
PhiX control
 17 Prepare a fresh 4 nM solution of PhiX from 10 nM stock or use previously diluted PhiX
solution if available.
 18 Combine 5 µL of the 4nM PhiX solution and 5 µL of the 0.1 N NaOH.
 19 Quick vortex and quick spin.
Protocol #06
 20 Incubate at room temperature for 5 minutes.
 21 Add 5 µL of 200 µM Tris-HCl, pH 7.0
 22 Quick vortex and quick spin.
 23 Add 985 µL of chilled Hybridization Buffer to the denatured library. Vortex before.
The total volume is 1 mL at 20 pM concentration.
 24 Quick vortex and quick spin.
 25 Transfer 35 µL of the diluted library to a new tube.
 26 Add 465 µL of chilled Hybridization Buffer. Vortex before use.
The total volume is 500 µL at 1.4 pM concentration.
Loading the MiniSeq
 27 Combine the library and the PhiX control. Quick vortex and quick spin.
The PhiX control should be added to the library so that its final concentration is between
5 and 15 %. E.g. combine 450 µL of the library and 50 µL of the PhiX.
 28 Pierce the aluminium cover on the cartridge at position 16 using a clean and empty
1 mL pipette tip. Use firm downward pressure. When pierced clean the edges.
 29 The resulting mixture of library and PhiX control is to be loaded in position 16 –
marked by: Load library here.
 30 Positions 24, 25 and 28 also need to be pierced for the addition of custom primers.
Use clean and empty 1mL for each position. Beware of splashback.
 31 Add the sequencing primers according to the table:
Primer Position Volume
Read 1 primer 24 3.3 µL
Read 2 primer 25 3.6 µL
Index 1 primer 28 4.9 µL
Index 2 primer 28 4.9 µL
 32 Mix the positions 24, 25 and 28 with a 1mL pipette set to 400 µL by pipetting up and
down. Make sure not to introduce an excess of bubbles.
 33 Check the cartridge from below. Knock out any bubbles present down in the wells.
 34 Remove the wash cartridge and insert the prepared sequencing cartridge.
 35 Take out a fresh flow cell. Check the flow cell for specks of dust or any other dirt.
Clean the flow cell if necessary. Be careful not to introduce any more dirt.
 36 Be careful when inserting a new flow cell not to crack or damage it.