Nextera XT Protocol
Nextera XT Library Preparation Cheat Sheet
Written by Steve Doyle (s.doyle@latrobe.edu.au) - Feb 2014
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Simplified version of Nextera XT protocol, which some modification
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Make sure to familiarize yourself with the full protocol and be aware of the
difference
Sample Setup for 24 wells (fill in the blanks)
- good practice to make a plate map with sample names clearly shown to ensure there is no
confusion, particularly during indexing and downstream bioinformatics analyses
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Nextera XT Protocol
STEP 1: Tagmentation of genomic DNA
Preparation
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Thaw on ice
o amplicon tagment mix (ATM)
o tagment DNA buffer (TD)
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neutralization buffer needs to be at room temperature
o vortex if precipitate is present
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invert all reagents 3-5 times, followed by pulse spin
1. label a 96-well PCR plate
2. add 10 ul of tagment DNA buffer to each well
3. add 5 ul of DNA (0.2ng/ul) to each well
4. add 5 ul of amplicon tagment mix to each well using multichannel pipette and
pipette up and down to mix
5. cover plate with plate seal
6. centrifuge at 280g at 20C for 1 min
7. put plate in PCR machine
a. 55C for 5 mins
b. hold at 10C
8. remove seal, and add 5 ul of neutralization buffer to each well
9. pipette up and down to mix
10. cover plate with plate seal
11. centrifuge at 280g at 20C for 1 min
12. incubate at room temperature for 5 mins
Nextera XT Protocol
STEP 2: PCR Amplification (starting volume: 25 ul)
Preparation
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thaw on bench (allow 20 mins)
o nextera PCR mastermix (NPM)
o index primers
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invert all reagents 3-5 times, followed by pulse spin
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Arrange index primers in the following order
White caps (i5)
S504 S503 S502 S501
Orange caps (i7)
N701 N702 N703 N704 N705 N706
1. Add 15 ul of nextera PCR mastermix to each well
2. Add 5 ul of index 2 primers (white caps) to each column and pipette up and down to
mix
a. Discard original caps and apply new caps provided with kit
3. Add 5 ul of index 1 primers (orange caps) to each row and pipette up and down to
mix
a. Discard original caps and apply new caps provided with kit
4. Discard original caps and apply new caps provided with kit
Nextera XT Protocol
5. cover plate with plate seal
6. centrifuge at 280g at 20C for 1 min
7. perform PCR
a. 72C – 3 mins
b. 95C – 30 secs
c. 12 cycles of
i. 95C – 10 secs
ii. 55C – 30 secs
iii. 72C – 30 secs
d. 72C – 5 mins
e. hold at 10C
8. centrifuge at 280g at 20C for 1 min
9. ***SAFE STOPPING POINT – PUT IN FRIDGE FOR UP TO 2 DAYS ***
STEP 3: PCR Cleanup (starting volume: 50 ul)
Preparation
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bring ampure XP beads to room temperature
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make 80% Ethanol – 400ul is needed per sample well – must be made fresh
1. add 30 ul of ampure XP beads to each well – this is a 0.6X ampure clean
2. transfer entire 50 ul sample to new round-bottom 96-well plate and pipette up and
down to mix
3. incubate at room temp for 10 mins
4. place plate on magnet for 5 mins (or until supernatant has cleared) – keep plate on
magnet for steps 4-9
5. aspirate supernatant and discard
6. carefully add 200 ul of 80% Ethanol to each well and wait 30 secs
7. aspirate supernatant and discard
8. repeat steps 6 & 7 for total of two washes
Nextera XT Protocol
9. check wells have no liquid left (must get rid of all EtOH) and allow to dry for ~ 5mins,
or until no EtOH can be detected
10. remove plate from magnet, and add 52.5 ul resuspension buffer to each well, and
pipette up and down to mix
11. incubate at room temperature for 5 mins
12. place plate on magnet for 5 mins (or until supernatant has cleared)
13. transfer 50 ul from each well into a new PCR plate
14. ***SAFE STOPPING POINT – PUT IN FREEZER FOR UP TO 7 DAYS ***
STEP 4: Library Normalisation and Pooling (starting volume: 50 ul)
***NOTE***: from here on, we are deviating from the Illumina protocol. This custom
protocol gives you more flexibility if further sequencing of select samples is required. It also
gives you much more control of the amount of DNA loaded onto the MiSeq.
1. Determine the concentration of each sample using the Qubit
Concentration of each sample (fill in the blanks)
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A
B
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D
E
F
G
H
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Nextera XT Protocol
2. Calculate the total DNA amount per sample (ie. ng/ul x volume = total ng)
3. Determine a suitable amount of DNA per sample that will allow you to normalize the
amount of DNA per sample and pool
a. Often the simplest way to do this is find the sample that has the lowest
amount of DNA, eg. 5 ng, and then determine the volume that you need to
take from every other sample that will also give you 5 ng.
b. If you have a lot of DNA, choose an arbitrary amount, and determine to
volume of each sample that will give you that amount
4. Once a suitable amount of DNA has been decided on, transfer the same amount to a
single microcentrifuge tube
a. NOTE – this is NOT equal volume per sample, but EQUAL molar
concentrations, where equal mass (ng) per sample is sufficient
5. OPTIONAL: if there is plenty of DNA, run a sufficient amount on a 2% agarose gel
with 100-bp ladder to check the size distribution of the library. If there is not enough
DNA to waste on a gel, assume the average fragment size is around 400-500bp
6. Adjust the concentration of the pooled DNA to 4 nM
a. This step is critical to the success of the sequencing run – accurate
concentration is key
b. Use Steve’s library dilution calculator to determine concentration of DNA in
tube
c. Use C1V1=C2V2 to adjust concentration to 4 nM
i. HINT: Assuming average frag size is 450bp, concentration should be
1.188 ng/ul
ii. Need minimum 5 ul or 5.94 ng total
7. Proceed to MiSeq loading guide