G4™ Sequencing Platform Quick Reference Card

Loading Concentration
10 pM 15 pM 20 pM 25 pM 30 pM

Prepare consumables

Thaw reagent cartridges at 2°C to 8°C for 2 days and equilibrate to room temperature for 30 minutes, or thaw in a room temperature water bath for 2.5 hours (wipe off residual water).

Remove flow cell from storage and equilibrate to room temperature for 30 minutes.

Create sample
data sheet
Optional

Download a sample data sheet template.

Fill out the Header, Settings, and Data sections.

Save the complete sample data sheet in the SampleData folder or in a location available to the instrument.

Denature and
dilute library

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.

If the starting library concentration is higher than 4 nM, dilute the library to 4 nM with TE (10 mM Tris-HCl, 1 mM EDTA, pH 8.0). Vortex and then centrifuge briefly.

Dilute the optional Singular PhiX control to 150 pM with TE. Vortex and then centrifuge briefly.

Denature the library and PhiX control in a separate new tubes. Vortex and then centrifuge briefly. For variations, see the Loading Concentration Calculator.

 

Sample Library (4 nM)

PhiX Control (150 pM)

Library

5 µL

5 µL

0.2 M NaOH

5 µL

5 µL

Incubate for 5 minutes at room temperature.

To the same tubes, add Tris-HCl to neutralize, then vortex to mix and centrifuge briefly.

200 mM Tris-HCl pH 7.0

5 µL

5 µL

To the same tubes, add water to dilute the sample library to 200 pM, then vortex to mix and centrifuge briefly.

Molecular-grade water

85 µL

-

Place the librarylibraries on ice until ready to proceed.

Dilute the 200 pM library and PhiX control in Sample Loading Buffer to the loading concentration in a new tube. Vortex and then centrifuge briefly. Loading concentration may differ for your application.

 

10 pM

15 pM

20 pM

25 pM

30 pM

Water

135 µL

127.5 µL

120 µL

112.5 µL

105 µL

Library, 200 pM

15 µL

22.5 µL

30 µL

37.5 µL

45 µL

2x Sample Loading Buffer

150 µL

150 µL

150 µL

150 µL

150 µL

PhiX control, 50 pM

-

-

-

-

-

TOTAL

300 µL

300 µL

300 µL

300 µL

300 µL

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

Prepare
sample cartridge

Obtain a sample cartridge for each flow cell.

Equilibrate the sample cartridge to room temperature for 30 minutes.

Remove the sample cartridge from the sealed bag. Avoid extended light exposure.

Pipette 260 µL diluted library into the appropriate well associated with each lane as listed in the sample data sheet.

Set aside until prompted to load sample cartridges onto the G4 instrument.

Set up
new run

To set up a new run, use the G4 Instrument Control Software on the instrument.

Specify Run Parameters

Select Sequence from the main menu, and then select +.

Assign a name for your run.

Specify the run type: Single or Paired.

Enter the number of cycles.

Specify if the run requires , single or dualIndex Reads.

If the run is indexed, enter the number of cycles for any index reads, 8 to 24 base pairs.

Select the forward arrow or scroll down to proceed.

Assign Sample Data

Select a category for your sample data: Now, or Not Required.

If you are assigning the sample data sheet now:

  1. Select the upload button and browse to the sample data sheets.
  2. Select the button to the right of each sample data sheet for the flow cell.
  3. Repeat step until you have added all sample data sheets you want to add.

Select the forward arrow or Continue to proceed.

Review Run Setup

Review run setup. Select the forward arrow to proceed.

Empty waste
and load
consumables

Empty the waste reservoir.

Invert reagent cartridges 5–10 times, then gently tap the cartridge on the bench surface.

Remove each flow cell from the packaging and remove the seals covering the ports and any seal adhesive.

When prompted load the wash buffer cartridges, flow cells, reagent cartridges, and sample cartridges.

Start
run

After the pre-run system check is complete, select Start Run.