pA-Tn5 Loading Protocol for Active Motif CUT&Tag-IT™ Assays
Recipe for 1X pA-Tn5 Transposome Storage Solution:
ChemicalFinal Conc.

Glycerol (Molecular Biology),density 1.28 g/mL

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

0.2%

EDTA 0.5 M, pH 8
CAS 6381-92-6

0.2 mM

Potassium Hydroxide
CAS 1310-58-3

pH to 7.2

Water, Molecular Biology GradeStore 1X pA-Tn5 Transposome Storage Solution at room temperature.
•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

For Research Use Only. Not for use in diagnostic procedures.

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pA-Tn5 Loading Protocol
Final formulation of pA-Tn5 in the loaded CUT&Tag-IT™ Assay Assembled Transposomes:
ChemicalFinal Conc.

Recombinant pA-Tn5 Transposase,bulk (Concentration will be on tube/paperwork – typically 0.3-0.4 mg/mL) MW 72.7 kDa

4.25 - 5.5 µM(dependenton starting
concentration)

1X Transposome Storage Solution

1X

Oligo A, 100 µM

6.0 µM

Oligo B, 100 µM

6.0 µM

Mosaic End Reverse Oligo, 100 µM

6.0 µM

Make Mosaic End Adapter Mixes A and B (note oligos are resuspended in TE):
Anneal Oligo A
1. Mix equal volumes of Oligo A (100 µM) with Mosaic End Reverse Oligo (100 µM).
2. Heat in a thermal cycler (95°C for 3 minutes, 65°C for 5 minutes, cool to 10°C (with a ramp rate of 0.1°C/second for all cooling steps).
Anneal Oligo B
1. Mix equal volumes of Oligo B (100 µM) with Mosaic End Reverse Oligo (100 µM).
2. Heat in a thermal cycler (95°C for 3 minutes, 65°C for 5 minutes, cool to 10°C (with a ramp rate of 0.1°C/second for all cooling steps).
Prepare CUT&Tag-IT Assay Assembled Transposomes (pA-Tn5 MW 72.7 kDa)
1. In one tube, combine pA-Tn5 in a 1:1 molar ratio with annealed primers.
For example, final concentration of 8 µM pA-Tn5, 4 µM Annealed Oligo A, and 4 µM Annealed Oligo B.
2. Vortex briefly (~1 second) on a low power setting or flick tube gently to mix.
3. Leave at room temperature for 50 minutes.
4. Store at -20°C.

For Research Use Only. Not for use in diagnostic procedures.

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