Nextera XT DNA Library Prep Kit

The protocol is optimized for 1 ng of input DNA (Small genomes, PCR amplicons or plasmids)
Before Starting Step Procedures Equations & Notes
80% ethanol Clean up PCR amplicon
(Freshly prepared) The PCR amplicon must be > 300 bp.
For 50 ml 1- Transfer 50 µl PCR product from each well of the PCR
40 ml Abs EtOH + 10 ml plate (The ratio of PCR product to volume of beads is 3:2).
dis H2O. 2- Shake well the Agencourt AMPure XP bottle before use.
Then add 30 µl AMPure XP beads to each well.
3- Mix reagent and sample thoroughly by pipette mixing 10
times.
4- Shake at 1800 rpm for 2 minutes.
5- Incubate at room temperature for 5 minutes.
6- Place on a magnetic stand and wait until the liquid is clear
(~2 minutes).
7- Remove and discard all supernatant from each well.
8- Wash two times as follows.
a. Add 200 µl fresh 80% EtOH to each well.
b. Incubate on the magnetic stand for 30 sec.
c. Remove and discard all supernatant from each well.
9- Using a 20 µl pipette, remove residual 80% EtOH from
each well.
10- Air-dry on the magnetic stand for 15 minutes.
11- Remove from the magnetic stand.
12- Add 52.5 µl RSB to each well.
13- Shake at 1800 rpm for 2 minutes.
14- Incubate at room temperature for 2 minutes.
15- Place on a magnetic stand and wait until the liquid is clear
(~2 minutes).
16- Transfer 50 µl supernatant from the midi plate to a new
Hard-Shell PCR plate
Qubit® dsDNA High DNA Quantification Signal is stable for 3
Sensitivity (HS) Assay kit 1- The Qubit™dsDNA HS assay requires 2 standards. hours.
2- Label the Qubit™ assay tube lids.
10 mM Tris HCl 3- When all solutions are at room temperature, make the
pH 7.5–8.5
Qubit™ working solution:
1M = MWg/mol in 1000 Qubit™ dsDNA HS reagent Qubit™ dsDNA HS buffer
ml 1 µl x No. of Samples 199 µl x No. of Samples
4- Load working solution into individual assay tubes so that For Dilution of Sample
10 mM = MWg/mol /100
the final volume in each tube after adding sample is 200 μL. use this equation:
in 1000ml
Or Standard tubes Sample tubes
10 mM = MWg/mol /10000 190 µl 10 µl Standard 198 µl 2 µl Sample C 1 V1 = C 2 V2
in 10ml
5- Mix by vortexing 2–3 seconds. Then incubate for 2 min at C1 = Initial
RT. concentration
6- Read Standards and Samples by Qubit Fluorometer. V1 = Initial volume
7- Dilute Samples to 0.2 ng/µl by 10 mM Tris HCl pH 7.5–8.5 C2 = Final concentration
V2 = Final volume
 Tagment Genomic DNA

gDNA Thaw on ice. 1- Add the following volumes in the order listed to each well
Invert the of a new PCR plate. Pipette to mix.
thawed tubes
ATM 3–5 times, and
then centrifuge TD 10 µl
TD briefly Normalized gDNA 5 µl
Check for ATM 5 µl
precipitates. If Pipette to mix.
present, vortex
NT until all 2- Centrifuge at 280 × g at 20°C for 1 minute.
particulates
3- Place on the preprogrammed thermal cycler and run the
are
resuspended.
tagmentation program.
55°C 10°C
Preheated lid
5 minutes Hold
When the sample reaches 10°C, immediately proceed to step
5 because the transposome is still active.
4- Add 5 µl NT to each well. Pipette to mix.
5- Centrifuge at 280 × g at 20°C for 1 minute.
6- Incubate at room temperature for 5 minutes. The PCR plate
contains 25 µl tagmented and neutralized gDNA, all of
which used in the next step.
Amplify Libraries
Index Only prepare The index adapters and Nextera PCR Master Mix are added
adapters adapters being
used. Thaw at directly to the 25 μl of tagmented gDNA from the previous step.
(i5 and i7) RT for 20 1- [24 libraries – for Example] Arrange the index adapters in
minutes. the TruSeq Index Plate Fixture as follows.
Invert each
tube to mix. 2- Arrange Index 1 (i7) adapters in columns 1–6.
Centrifuge 3- Arrange Index 2 (i5) adapter in rows A–D.
briefly. 4- Add 5 µl of each Index 1 (i7) adapter down each column.
NPM Thaw on ice Replace the cap on each i7 adapter tube with a new orange
for 20 cap.
minutes.
5- Add 5 µl of each Index 2 (i5) adapter across each row.
Microseal 'A' film
Replace the cap on each i5 adapter tube with a new white
New orange & white cap.
caps. 6- Add 15 µl NPM to each well containing index adapters.
Pipette to mix.
7- Centrifuge at 280 × g at 20°C for 1 minute.
8- Place on thermal cycler and run the PCR program.
72°C 3 minutes 1
95°C 30 seconds 1
95°C 10 seconds
55°C 30 seconds 12 Cycles
72°C 30 seconds If you are stopping, seal
the plate and store at
72°C 5 minutes 1
2°C to 8°C for up to
10°C Hold
2 days. Alternatively,
The volume is 50 µl. leave on the thermal
cycler overnight.
SAFE STOPPING POINT

Preparing the Gel-Dye
Mix:
1- Allow the blue- capped
High Sensitivity DNA dye
concentrate and red- capped
High Sensitivity DNA gel
matrix to equilibrate to room
temperature for 30 minutes.
2-Vortex the blue- capped
vial with High Sensitivity
DNA dye concentrate (blue)
for 10 seconds and spin down.
Make sure the DMSO is
completely thawed.
3-Pipette 15 µl of the blue-
capped dye concentrate
(blue) into a red- capped
High Sensitivity DNA gel
matrix vial (red). Store the
dye concentrate at 4 °C in the
dark again.
4-Cap the tube, vortex for 10
seconds.
5-Transfer the complete gel-
dye mix to the top receptacle
of a spin filter.
6-Place the spin filter in a
microcentrifuge and spin for
10 minutes at room
temperature at 2240 g ± 20 %
(for Eppendorf
microcentrifuge, this
corresponds to 6000 rpm).
7-Discard the filter according
to good laboratory practices.
Label the tube and include the
date of preparation.
Starting the 2100 Expert
Software
Clean Up Libraries
Clean up libraries by AMPure XP beads as Step 1 If you are stopping, seal
the plate and store at
-25°C to -15°C for up to
SAFE STOPPING POINT seven days.
Library Quantification
Quantification of libraries by Qubit as Step 2
Check Libraries
-The prepared gel-dye mix is
Fragment size detection by Bioanalyser using a High Sensitivity sufficient for 5 chips. Use the
DNA Kit. gel-dye within 6 weeks of
preparation.
Loading the Gel-Dye Mix - Protect the gel-dye mix
1- Allow the gel-dye mix to equilibrate to room temperature for 30 from light. Store the gel-dye
min before use. mix at 4 °C when not in use
2- Put a new High Sensitivity DNA chip on the chip priming for more than 1 hour.
station.
3- Pipette 9 μL of gel-dye mix in the well marked .
4- Make sure that the plunger is positioned at 1 mL and then close
the chip priming station.
5- Press plunger until it is held by the clip.
6- Wait for exactly 60 s then release clip.
7- Wait for 5 s, then slowly pull back the plunger to the 1 mL
position.
8- Open the chip priming station and pipette 9 μL of gel-dye mix in
the wells marked G.
Loading the Marker
1- Pipette 5 μL of marker (green) in all sample and ladder wells.
Do not leave any wells empty.
Loading the Ladder and Samples
1- Pipette 1 μL of High Sensitivity DNA ladder (yellow) in the well
marked .
2- In each of the 11 sample wells pipette 1 μL of sample (used
wells) or 1 μL of marker (unused wells).
3- Put the chip horizontally in the adapter and vortex for 1 min at
the indicated setting (2400 rpm).
4- Run the chip in the Agilent 2100 Bioanalyzer instrument
within 5 min.
Always insert the pipette tip
to the bottom of the well
when dispensing the liquid.
5- Select AssaysdsDNAHigh Sensitivity DNA Placing the pipette at the
6- Click the Start. edge of the well may lead to
poor results.
Checking Results:

1- High Sensitivity DNA Ladder Well Results

To check the results of your run, select the Gel or
Electropherogram tab in the Data context. The electropherogram
of the ladder well window should resemble to those shown below.

Major features of a successful ladder run are:

• 15 peaks for High Sensitivity DNA ladder (including markers).
• All peaks are well resolved.
• Flat baseline.
• Correct identification of both markers.

2- High Sensitivity DNA Sample Well Results

select the sample name in the tree view and highlight the Results Major features for a
successful High Sensitivity
sub- tab. The electropherogram of the sample well window should DNA sample run are:
resemble the one shown here. • All sample peaks appear
between the lower and upper
marker peaks. If some sample
peaks are outside the marker
bracket, adjust the upper or
lower marker.
• Flat baseline
• Baseline readings at least 5
fluorescence units (see Zero
Baseline in the
• Marker readings at least 3
fluorescence units higher
than baseline readings.
• Both marker peaks well
resolved from sample peaks
(depends on sample).

Beast Result between 600 :

800 bp

PCR grad water
Thawing the reagent
cartridge before denaturing
and diluting libraries.
Prepare 1N NaOH
Prepare a Fresh Dilution
of NaOH
NaOH (0.2N)
1- Combine the following
volumes in a
microcentrifuge tube.
 Laboratory-grade water
(800 µl).
 Stock 1.0 N NaOH
(200 µl) the result is 1 ml
of 0.2 N NaOH.
2- Invert the tube several
times to mix.
Prepare HT1
1- Remove HT1 from -25°C
to -15°C storage and thaw
at room temperature.
2- Store at 2°C to 8°C until
you are ready to dilute
denatured libraries.
Preheat the incubator to
98°C.
10 mM Tris-Cl, pH 8.5
with 0.1% Tween 20
Calculation of dsDNA library concentration
Converting ng/µl to nM when calculating dsDNA library
concentration
1. Determine the average size of the library by running it on an For Dilution of Sample use
Agilent Technologies 2100 Bioanalyzer. this equation:
2. Use the following formula to convert from ng/μl to nM:
C1 V1 = C2 V2
C1 = Initial concentration
V1 = Initial volume
C2 = Final concentration
V2 = Final volume
Dilute each sample to (2 nM or 4 nM) using PCR grad water.
Pool Libraries
Pooling libraries combines equal volumes of normalized libraries in
a single tube.
Denaturation & Dilution of Libraries
Weight= NEV÷1000
Denature a 4 nM Library Denature a 2 nM Library Where ,
 4 or 2 nM library (5 µl). N= normality
E= equivalent weight
 0.2 N NaOH (5 µl).
V= volume
 Vortex briefly and then centrifuge at 280 × g for 1 minute.
To make NaOH (0.2N)
 Incubate at room temperature for 5 minutes.
500 ml
 Add 990 µl prechilled HT1 to the tube containing denatured (0.2×39.99×500)÷ 1000
library. =3.99g
The result is 1 ml of a 20 pM The result is 1 ml of a 10 pM
denatured library. denatured library. Use the fresh dilution
NaOH within 12 hours.
Denature Diluted Library
1- Place the tube on the preheated incubator for 2 minutes.
2- Immediately cool on ice.
3- Leave on ice for 5 minutes.
Dilute Denatured 20 or 10 pM Library
Concentration 8 pM Concentration 8 pM
20 pM library 240 µl 10 pM library 480 µl
Prechilled HT1 360 µl Prechilled HT1 120 µl
Invert to mix and then pulse centrifuge then leave on ice until use.
Denaturation & Dilution of Phix control
MiSeq Reagent Kit v3 MiSeq Reagent Kit v2
Dilute the denatured PhiX Dilute the denatured PhiX
control to 20 pM control to 12.5 pM
1- Combine the following volumes in a microcentrifuge tube. You can store the denatured
 10 nM PhiX library (2 µl). 20 pM PhiX library up to 3
weeks at -15°C to -25°C.
 10 mM Tris-Cl, pH 8.5 with 0.1% Tween 20 (3 µl)
 0.2 N NaOH (5 µl)
2- Vortex briefly to mix.
3- Centrifuge at 280 × g for 1 minute.
4- Incubate at room temperature for 5 minutes.
 5- Prechilled HT1 (990 µl).
The result is 1 ml of a 20 pM PhiX library then Invert to mix.
Dilute Denatured PhiX to 12.5 pM
 20 pM denatured PhiX library (375 µl).
 Prechilled HT1 (225 µl).
The result is 600 µl of a 12.5 pM PhiX library then Invert to mix.
Combine Library and PhiX Control
Combine the following volumes of denatured PhiX control and
denatured library.

Denatured and diluted PhiX 6 µl

Denatured and diluted library 594 µl