Irys NLRS DNA labeling and Data collection
Equilibrate DNA ~ 30 min at room temp .
Pipet mix 3x with wide bore tip .
Prepare nicking master mix One 10μL rxn : 1μL buffer + DNA + enzyme in 200μL thin - wall PCR tube .
Blood BspQI : 5 - 7u ( BNG3 ) Cell line BspQI : 3 - 5u ( BNG3 ) Other BspQI : 5 - 10u ( BNG3 ) BbvCI : 4 - 8u ( BNG2 ) Q + C : 3 / 2 , 6 / 4 , 9 / 6u ( BNG2 ) .
Mix 4x with Xplorer Plus Pipettor set to 8μL at lowest speed .
Pulse spin 2sec .
Incubate in a thermal cycler for 2 hrs at 37°C with heated - lid . ( For the rest of the protocol , protect from light ) .
Prepare the Labeling Master Mix .
reagentamount10x labeling buffer1.5μL10x labeling mix1.5μLTaq 5u / μL1μLH201μL .
Add 5μL Labeling Master Mix .
Mix 4x with Xplorer Plus Pipettor set to 13μL at lowest speed .
Pulse spin 2sec .
Incubate in a thermal cycler for 60 min at 72°C with heated - lid .
Prepare the Repair Master Mix .
reagentamount10x Thermo Pol0.5μL50x Repair Mix0.4μL50mM NAD + 0.4μLTaq DNA ligase1μLH202.7μL .
Mix 4x with Xplorer Plus Pipettor set to 18μL at lowest speed .
Pulse spin 2sec .
Incubate in a thermal cycler for 30 min at 37°C with heated - lid .
Place NLR reaction on ice .
Add 1μL Stop Solution .
Mix by gently stirring 5 times with the pipet tip .
Pulse spin 2sec .
Add 5μl of the Repair Master Mix .
Equilibrate Staining Master Mix components to room temp .
Prepare the Staining Master Mix .
reagentamount4x flow buffer15μL1M DTT12μLDNA stain1.5μLH2O11.5μL .
Aliquot 40μL of the Staining Master Mix into a 0.6mL amber tube .
Transfer NLR to Staining Mix with standard tip .
Pulse spin 2 sec .
Gently mix NLRS DNA 5x with wide bore tip set to 50μL .
Pulse spin 2 sec .
Place staining reactions at 4°C overnight to ensure uniform DNA staining .
Equilibrate NLRS to room temp for ~ 30 min .
Gently mix DNA 3x with wide bore tip set to 50μL .
Pulse spin 2 sec .
Remove 2μL aliquots from the Top , Middle and Bottom to separate Qubit Assay tubes .
Add 18uL of Qubit HS Buffer .
Sonicate 10 min in a bath sonicator .
Add 180μL Qubit reagent mix .
Vortex 5 seconds .
Pulse spin 2 sec .
Incubate at least 2 min at room temp .
Quantitate with Qubit 2.0 Fluorimeter .
NLRS DNA conc : 3 - 10ng / μL .
CV < 25 % .
Load sample into Irys chip and start data collection with a 30 - cycle run on irys system
