Tn5 Loading Protocol
Quick Guide

Catalog No. 81286, 81284
vA1
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Overview
Protein Details
Recombinant Tn5 Transposase protein was expressed in E. coli as the full-length protein (Accession No.
ADY68344.1) without any tags or redundant amino acids. This Tn5 mutant is called ‘hyperactive Tn5 transposase’
since it can be used to randomly insert Tn5 Transposon into any target DNA in vitro. The molecular weight of thisprotein is 53.3 kDa.
Diagram
LegendMosaic End Reverse SequenceOligo B

Oligo A

Genomic (sample) DNA

1.

Recombinant Tn5

Mixing and Annealing Oligo A and Oligo B with the reverse sequence (R) of the Mosaic End Sequence(ME), in two separate reactions.

MER- Oligo A

MER- Oligo B

2.

Loading the transposomes with the annealed oligos.

3.

Tagmentation reaction

4.

Loading efficiency test

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Before Starting
1.

Order the oligos needed to load the transposomes.
• Oligo A: 5’ TCG TCG GCA GCG TCA GAT GTG TAT AAG AGA CAG• Oligo B: 5’ G TCT CGT GGG CTC GGA GAT GTG TAT AAG AGA CAG• Mosaic End Reverse Oligo: 5’ PO4-CTG TCT CTT ATA CAC ATC T

Note:

The sequence in bold will anneal with the Mosaic End Reverse Oligo. The underlined sequenceof the Oligo A is the Nextera Read 1 Sequencing Primer (Rd1 SP) and Oligo B is the Nextera
Read 2 Sequencing Primer (Rd2 SP).

Resuspend the Oligo A, Oligo B and Mosaic End Reserve Oligo with 10 mM Tris pH8 to a final concentration of100 μM.
2.

Prepare the 1X Tn5 Transposome Storage Solution. Store it at room temperature.
Chemical

CAS Number

Glycerol (Molecular Biology), density 1.28 g/mL

Final Concentration
50% (v/v)

Sodium chloride (NaCl) 5 M

CAS 7647-14-5

200 mM

HEPES, 1 M, pH 7.5

CAS 7365-45-9

100 mM

Dithiothreitol (DTT)CAS 3483-12-3

2.0 mM

Triton X-100

CAS 9002-93-1

0.1%

EDTA 0.5 M, pH 8

CAS 6381-92-6

0.2 mM

Potassium Hydroxide

CAS 1310-58-3

pH to 7.2

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Tn5 Loading Protocol
Make Mosaic End Adapter Mixes A and B:
1.

Calculate the molarity of the transposomes. The concentration will be on the vial or on the Technical Data
Sheet (TDS) provided, and is typically 0.3-0.5 mg/mL, while the molecular weight is 53.3 kDa.
For example:• Concentration: 0.5mg/ml or changing the units to µg/µL: 0.5 µg/µL• MW: 53.3 kDa or 53300 daltons
Molarity (μM) = (Concentration (µg/µL) / MW (kDa)) * 1000
Molarity (μM) = (0.5 /53.3) * 1000= 9.38 μM
In this example, the molarity will be 9.38 μM

2.

Combine equal volumes of Mosaic End Reverse Oligo (100 µM) and Oligo A (100 µM). The total volumeof this reaction is arbitrary and is dependent on the final quantity of loaded transposase needed. Refer toStep 8 below to see the volume required in the example scenario from Step 1.

3.

Combine equal volumes of Mosaic End Reverse Oligo (100 µM) and Oligo B (100 µM).

Note:

The resulting molarity of this annealing reaction is 50 µM. The annealing of the Mosaic End
Reverse Oligo with the Oligo A or Oligo B will reduce the number of molecules by half, bringing
the final molarity to 50 µM.

4.

Place tubes in a 95°C thermal cycler with heated lid (105°C) for 3 minutes.

5.

Stop the program and allow the mixed oligos to cool down to room temperature in the heat block without
opening the lid for 30 minutes.

6.

Remove samples from the heat-block and incubate at room temperature for an additional 30 minutes.

Prepare Assembled Transposomes
7.

In one tube, combine Tn5 in a 1:0.5:0.5 molar ratio with the annealed oligos. The annealed oligos are at aconcentration of 50 µM Oligo A-mosaic and 50 µM Oligo B-mosaic.
Example for loading the entire 10 µg Tn5 vial:
Starting Material:
Tn5 Molarity: 9.38 μM
Each annealed oligos Molarity: 50 μM
To achieve 1:0.5:0.5 molar ratio, each oligo is added at a molarity of 4.69 μM (half of 9.38 μM).

Note:

The final volume (Vf) of this reaction is arbitrary. If all 10 µg of the vial is to be loaded, 20 µL of
the Tn5 will be used in this reaction example (10 µg format for a concentration of 0.5 µg/µL):
Ci * Vi = Cf * Vf50 µM * Vi = 4.69 μM * 20 µL
Vi = 1.87 µL

In this example, add 1.87 µL Oligo A- mosaic and 1.87 µL Oligo B- mosaic to 20 µL Tn5 enzyme. Mix by
gently vortexing or by pipette.
Caution: This step is critical for proper transposomes loading. The Tn5 buffer contains 50% glycerol andis viscous. To mix the transposomes properly, flick the tube gently, or lightly vortex.

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8.

Incubate for 1 hour at room temperature. At this step, the loaded transposomes have a molarity of ~8 μM
in this example.

Note:
9.

A loading efficiency test can be performed now. Further dilution of the loaded Transposomes
is optional.

Dilute to 1 μM in 1x Transposome Storage Buffer. Add 166.18 µL of 1x Tn5 Transposome Storage Solution
to the 23.74 µL from the previous step to bring the loaded Tn5 to 1 μM. The final volume of this dilution is189.92 µL.

10. Mix gently by flicking the tube or pipetting up and down.
11.

Store at -20°C

12. Proceed with the loading efficiency test.
Note:

A loading efficiency test to assess the proper loading of the transposomes is useful todetermine the amount of loaded Tn5 needed to tagment 150 ng of genomic DNA.

Final formulation of diluted Tn5 in the loaded Assembled Transposomes
Final loaded anddiluted concentrations

Initial Concentrations
Recombinant Tn5 Transposase, bulk

1 μM

Oligo A, 100 μM in 10mM Tris-pH80.5 μM

Oligo B, 100 μM in 10mM Tris-pH80.5 μM

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1 μM

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Efficiency Test Loaded Tn5
You will need:• Assembled Tn5 Transposomes• Tagmentation Reaction Buffer• Genomic DNA, 50 ng/ μL• Freshly prepared 0.5% SDS• Thermal cycler• Columns or beads for purification• Agilent TapeStation or similar
Note:

Prepare Assembled Tn5 Transposomes and Tagmentation Reaction Buffer before starting.

1.

Prepare a master mix with Tagmentation Reaction Buffer and genomic DNA (keep genomic DNA constantat 150 ng and titrate volume of Assembled Tn5 Transposomes).

2.

Aliquot each sample and add remaining water to bring to volume (25 µL minus the Assembled Tn5
Transposomes volume).

3.

Add Assembled Tn5 Transposomes (for example, titrate 1 µL, 2 µL, 4 µL).
2x
Tagmentationbuffer

150 nggenomic DNA

Loaded Tn5

H2OTotalReaction 1103

1

620

Reaction 21032

520

Reaction 310334204.

Mix the reaction by pipetting up and down and place at 55°C for 7 minutes.

5.

Add 2.5 μL of 0.5% SDS and incubate for another 3 minutes at 55°C.

6.

Purify the tagmented DNA. This can be achieved by using any DNA column purification kit or a 1.2X
beads purification. For a 1.2X beads purification, add 27 µL of SPRI beads to the 22.5 µL final reaction
volume after step 5. Elute in 10 µL 10 mM Tris, pH 8.0.

7.

QC: Run 1 or 2 µL on Agilent TapeStation.

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Tn5 Tagmentation Results
Successful tagmentation

Unsuccessful tagmentation
Because there is no fragmentation of the genomic input DNA after tagmentation, this reaction isconsidered unsuccessful

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More assembled Tn5 is needed.
Both profiles correspond to successfully loaded Tn5 (no input DNA visible on the high molecular size end of theTapeStation) with slightly lower activity. More Tn5 should be used in subsequent reactions.
Performing a titration of the loaded Tn5 is helpful to assess tagmentation activity.

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Problem/Question

Recommendation

Why are the Oligos and theMosaic End Reverse Oligo
reconstituted in 10mM Tris pH8?

They could also be resuspended in low EDTA TE (10 mM Tris-HCl pH 8.0
0.1 mM EDTA) or molecular grade water. A high concentration of EDTA
may impact the performance of loaded Tn5 transposase.

If my trace doesn’t display
tagmentation, what should I
do next?

Try a higher volume of your loaded Tn5 per reaction. Or consider loading
again if there is Tn5 remaining. Even if there is little activity you will
still see a smear, but the fragments will be larger. If the unsuccessful
tagmentation trace is observed, it is likely that loading didn’t work.

Why didn’t my loading work?

The oligos and Tn5 likely were not mixed thoroughly enough. The Tn5
contains 50% glycerol, making mixing more difficult. Make sure to gently
vortex or mix thoroughly by slowly pipetting the solution up and down.

Can I use concentrated loadedtransposase, or do I need todilute it to 1 µM?

Test the concentrated loaded transposase and determine the amountof loaded transposase needed for the specific experimental plan.
Then, dilute the assembled Tn5 transposomes, or store them in theconcentrated form.

Why is the final concentrationof the annealed oligos
0.58 μM in the 1 μM loadedtransposase solution?

4.69 μM of each annealed oligo were added, and the final volume ofthe loading reaction is 23.74 µL. After dilution to 1 μM, it is a final volumeof 189.92 µL, and using the following equation, the molarity of eachannealed oligo can be calculated:
Ci * Vi = Cf * Vf
4.7 µM * 23.74 µL = Cf * 189.92 µL
Cf = 0.58 µM

Why is the final
concentration of the loaded
Transposomes ~8μM?

In the loading reaction, we add 20 µL of the recombinant Tn5
Transposase at a molarity of 9.38 µM. After the addition of each Oligo,
the final volume of the reaction is 23.6 µL. The following formula can beused to calculate the molarity of the loaded transposase:
Ci * Vi = Cf * Vf
9.38 µM * 20 µL = Cf * 23.74 µL
Cf = 7.90 μM ~ 8 μM

What is Oligo A, Oligo B andMosaic End Repair?

The “Oligo A” has a part of the Mosaic End Reverse Oligo sequence thatwill allow the annealing with the Mosaic End Reverse Oligo followed by
the Read 1 Sequencing Primer sequence (Rd1 SP). The “Oligo B” also
has a part of the Mosaic End Reverse Oligo sequence and the Read 2Sequencing Primer (Rd2 SP).
The reverse Mosaic End Sequence is the complementary sequence tothe Mosaic End Sequence on Oligo A and B. It is a 19 basepair sequencethat anneals to the 19 base pair ME region of Oligo A and Oligo B during
the annealing step, making that region double stranded. Tn5 monomers
bind this double stranded sequence and subsequently dimerize, resulting
in a “loaded” Tn5 transposome dimer.

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Recommendation

Can I use my own adapter
sequences? If so, will theprotocol need to be adjusted?

Yes. Adapter sequences will need to contain part of the Mosaic End
Reverse sequence (the bolded sequence in step one) to be annealed
to the Mosaic End Reverse Oligo. Longer adapters (up to 70 bp) resultin less efficient tagmentation. More transposomes may be required forthe reaction.

Can I use nuclei instead ofgenomic DNA?

Yes, nuclei can be used instead of the genomic DNA on the loading
efficiency test. The nuclei have to be permeable to allow the entrance ofthe loaded Transposomes. For reference, 150 ng of DNA corresponds to25,000 cells.

Where can I find
Tagmentation buffer?

2x Tagmentation Buffer is available in the ATAC-Seq Buffer Set (Cat. No.
53153) or several recipes can be found in the literature.

Do the Loaded Transposomes
act on single or double
stranded DNA?

The loaded transposomes will only tagment double stranded DNA andDNA-RNA hybrids.

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