Enabling Epigenetics Research

miRNA Target IP Kit
Catalog No. 25500(Version B1)

Copyright 2020 Active Motif, Inc.

Revision

Date

Description of Change

B1

August, 2024

BSA is no longer provided as a powder.
It is provided suspended in Wash Buffer AM7. See instructions forstoring after first thaw.
Updated formatting.

Information in this manual is subject to change without notice and does not constitute a
commitment on the part of Active Motif, Inc. It is supplied on an “as is” basis without any
warranty of any kind, either explicit or implied. Information may be changed or updated inthis manual at any time.
This documentation may not be copied, transferred, reproduced, disclosed, or duplicated,
in whole or in part, without the prior written consent of Active Motif, Inc. This documentation
is proprietary information and protected by the copyright laws of the United States andinternational treaties.
The manufacturer of this documentation is Active Motif, Inc.
© 2020 Active Motif, Inc., 1914 Palomar Oaks Way, Suite 150; Carlsbad, CA 92008. All
rights reserved.
All trademarks, trade names, service marks or logos referenced herein belong to theirrespective companies

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Contents
Overview  . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1
Flow Chart of Process . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .  2
Principle of the Method and Data . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .  3
Kit Components and Storage  . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .  4
Additional Materials Required . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .  5
Experimental Set Up . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .  6
Experimental Design  . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .  6
Growth of Cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .  7
Buffer Preparation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8
Quick Chart for Preparing Buffers  . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10
Protocols  . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11
Section A: miRNA Transfection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11
Section B: Lysate Preparation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12
Section C. Antibody Conjugation to Protein G Beads . . . . . . . . . . . . . . . . . . . . . . . 12
Section D. RNA Complex Immunoprecipitation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13
Section E. Proteinase K Digestion and RNA Purification . . . . . . . . . . . . . . . . . . . . . 14
Section F. Analysis of Samples by qRT-PCR . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19
Appendix . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20
Section G. Troubleshooting Guide . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20
Section H: Recommendations for Primer Design  . . . . . . . . . . . . . . . . . . . . . . . . . 22
Section I: Data Analysis  . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .  23
Section J. Use of Magnetic Beads and Included Bar Magnet . . . . . . . . . . . . . . . . 23
Technical Services . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .  27

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Overview
MicroRNAs (miRNAs) are a class of small, non-coding RNA molecules that act as posttranscriptional regulators of gene expression by binding to the 3-prime Untranslated Region(3´UTR) of messenger RNA transcripts (mRNAs). Most commonly, the interaction of a miRNA
with a 3´UTR results in either transcript degradation or inhibition of translation.
miRNA regulation plays a large role in many different biological processes, including
development, proliferation and differentiation. Changes in miRNA expression levels havebeen found in a variety of cancers, as well as in cardiac disease and neurological disorders,making miRNA-mediatedregulation an important topic in biomedical research.
Over 1,000 different miRNAs have been found in the human genome. Because they bindwith only partial complementarity, most miRNAs target multiple mRNA transcripts (up to 100
in some cases). In addition, individual mRNAs frequently contain binding sites for more than
one miRNA, and miRNA families that differ by as little as a single base pair have been found.
This creates a complex regulatory network that can act in a tissue and cell-state dependent
manner. As a result, it has been estimated that the expression of up to 60% of mammalianproteins may be regulated by miRNAs.
The targeting of a miRNA molecule to a specific mRNA is mediated through the formationof an RNA Induced Silencing Complex (RISC). Key components in the RISC complex are an
Argonaute protein (Ago) and a miRNA. The Ago protein binds the miRNA, positioning it in a
conformation that enables the RISC complex to base-pair in a Watson-Crick manner with amRNA transcript. The sequence of the miRNA molecule contained in a RISC complex enables
the precise targeting of specific mRNA transcripts.
Active Motif’s miRNA Target IP Kit utilizes protein G magnetic beads and a pan-Ago antibody
that recognizes Ago 1, Ago 2 and Ago 3 proteins to validate the actual physical interactionsof a miRNA with endogenous mRNA transcripts. It has been validated in both human andmouse cell lines.
The kit supplies sufficient reagents to perform 10 miRNA Target IP reactions using theincluded Ago1/2/3 Antibody and 10 negative control IP reactions using the included Negative
Control IgG. Following their immunoprecipitation (IP) as part of the RISC complex, themRNA transcripts are purified and amplified by qRT-PCR using gene-specific primers. A foldinduction is then calculated using the Ago1/2/3 Antibody and Negative Control IgG data,
which will indicate if the mRNA transcripts that were amplified were bound by the miRNA ofinterest. It should also be possible to profile the immunoprecipitated mRNA molecules using
conventional expression microarrays or RNA-Seq.

Product

FormatCatalog No.

miRNA Target IP Kit

10 rxns

25500

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1Flow Chart of Process

Cycle

Immunoprecipitate miRNA/mRNAcomplexes using the Ago1/2/3
antibody and protein G
magnetic beads (in duplicate).

∆ Rxn

Immunoprecipitate miRNA/mRNAcomplexes using the Negative
Control IgG and protein G
magnetic beads (in duplicate).

∆ Rxn

∆ Rxn

Immunoprecipitate miRNA/mRNAcomplexes using the Ago1/2/3
antibody and protein G
magnetic beads (in duplicate).

Grow cells and transfect with a non-targeting control miRNA.

Immunoprecipitate miRNA/mRNAcomplexes using the Negative
Control IgG and protein G
magnetic beads (in duplicate).

∆ Rxn

Grow cells and transfect with a miRNA of interest.

Cycle
Cycle
Cycle
After purifying RNA from the differrent IP reactions, perform qRT-PCR using gene-specific qPCR primer set(s) designed toamplify mRNA transcript(s) you believe will be bound by, and thus immunoprecipitated with, the miRNA of interest.

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2

Principle of the Method and Data
The targeting of a miRNA to a specific mRNA is mediated through the formation of an RNA
Induced Silencing Complex (RISC). While RISCs can contain a combination of different
RNA-binding proteins, at a minimum a RISC is comprised of an Argonaute protein (Ago) anda miRNA. The Ago protein binds the miRNA in a manner that enables the miRNA-loaded
RISC complex to base-pair with a mRNA transcript. The miRNA Target IP Kit utilizes a panAgo antibody and protein G-coupled magnetic beads to immunoprecipitate miRNA/mRNAcomplexes that have associated with Ago1, Ago2, or Ago3. This enables one to validate
which mRNA transcripts were bound by a specific miRNA.
Ribosome

ORF

5´
mRNA

3´UTR
Ago

AAA-3´
miRNA

RISC Complex
Figure 1: Translational repression by a miRNA-directed, RISC Complex.
The key components in a RISC Complex are an Argonaute protein (Ago) and a miRNA. The Agoprotein binds the miRNA, positioning it in a conformation that enables the RISC to base-pair in a
Watson-Crick manner with a mRNA transcript. This leads to either inhibition of translation (shown) or
increased degradation of the targeted transcript.

14

miR-122
Non-targeting

Fold Enrichment

12
10
8
6
4
2
0

G6PC3

SLC1A5GAPDH

Figure 2: miR-122 targets G6PC3 and SLC1A5.
The miRNA Target IP Kit was used on samples of HT1080 cells that had been transfected witha miR-122 mimic or a non-targeting miRNA control for 8 hours. Following IP using the Ago1/2/3
antibody or Negative Control IgG included in the kit, qRT-PCR was performed on the samples using
primers for G6PC3 and SLC1A5, which are known targets of miR-122, and for GAPDH, a common
housekeeping gene that is not known to be targeted by miR-122

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3Kit Components and Storage
Please store each component at the temperature indicated in the table below. Do not
re-freeze the Protein G Magnetic Beads. Once thawed, Protein G beads should be stored
at 4°C
Reagents

Quantity

Storage

Ago1/2/3 antibody (mAb) (1.0 µg/µL)

50 µL

-20°C

1 M DTT

100 µL-20°C

RNase Inhibitors

2 x 60 µL-20°C

Protease Inhibitor Cocktail (PIC)

2 x 100 µL-20°C

Ribonucleoside Vanadyl Complex

55 µL

-20°C

BSA Solution (50 mg/mL)

4 mL

-20°C

Proteinase K (10 µg/µL)

560 µL-20°C

Precipitation Buffer

1.5 mL-20°C

Glycogen (20 µg/µL)

40 µL

-20°C

IGF2 Primer pair (2.5 µM each)

90 µL

-20°C

Protein G Magnetic Beads*

1 mL

4°C

10X PBS

100 mL

4°C

Negative Control Mouse IgG (0.2 µg/µL)

250 µL

4°C

Lysis Buffer AM6

5 mL

4°C

10X Wash Buffer AM7

20 mL

4°C

0.5 M EDTA

1 mL

RT

10% SDS500 µL

RT

Salt Solution

500 µLRT

Bar magnet

1 magnet

RT

Glue dots

2 sheetsRT* The Protein G Magnetic Beads are shipped on dry ice, and can be stored frozen until their firstuse. However, Protein G beads should not be re-frozen by the customer. After they have been
thawed the Protein G Magnetic Beads should be stored at 4ºC.

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4Additional Materials Required•

RNase-free tubes (PCR and microcentrifuge)•

RNase-free filtered pipette tips and pipettor•

RNase-free water•Rocking platform for culture plates•

Cell scraper (rubber policeman)•

15 and 50 mL conical tubes•

Microcentrifuge (table top centrifuge 4°C)•

Freezer capable of -80ºC storage•

Apparatus to rotate tubes end-over-end at 4°C (e.g. a Labquake from Barnstead/
Thermolyne with a tube holder for microcentrifuge tubes)•

Vortex•Heat block or thermo-mixer•

Phenol:chloroform:isoamyl alcohol (25:24:1) (e.g. Fisher Scientific AC327115000)•

Chloroform•

100% ethanol (absolute)•

80% ethanol•

Thermal cycler•

miRNA Mimics and non-targeting miRNA Mimics Active Motif offers over 800 different
human miRNA Mimics and non-targeting miRNA Mimics. Please go to activemotif.com/
mimics for more information.

•

DharmaFECT 4 Transfection Reagent (e.g. Dharmacon Catalog No. T-2004-01), or areagent known to work well for miRNA transfection in your specific cell type•(Optional) 2-Step Reverse Transcription Kit (e.g. Applied Biosystems High-Capacity
RNA-to-cDNA Kit, Applied Biosystems Catalog No. 4387406)•(Optional) Gene-specific qPCR primer pairs for enrichment analysis; see Sections
F&H•(Optional) SYBR Green qPCR master mix (e.g. Bio-Rad Catalog No. 170-8882)

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5Experimental Set Up
PLEASE READ THE ENTIRE PROTOCOL BEFORE STARTING!

Experimental Design
This kit was designed to validate if specific mRNA transcripts are bound by a particular
miRNA. Therefore, you will transfect cells with your miRNA of interest and then perform
immunoprecipitation (IP) using a pan-Ago antibody that recognizes Ago1, Ago2 and Ago3.
This will pull down miRNA/mRNA complexes that have associated as part of a RISC complex;
IP will also be
performed with a Negative Control IgG.
After purifying the RNA and making cDNA, you will use gene-specific qPCR primer
set(s) designed to amplify mRNA transcript(s) you believe will be bound by, and thus
immunoprecipitated with, the transfected miRNA of interest. The Ago1/2/3 Antibody and
Negative Control IgG data will then be used to calculate a Fold Enrichment; if a specificmRNA was bound by the miRNA, more copies of that transcript will be pulled down by
the pan-Ago IP relative to the Negative Control IgG. As a negative control you should
also perform pan-Ago and Negative Control IgG IP experiments on cells that have beentransfected with a non-targeting control miRNA to be certain the effect seen with the miRNAof interest is not due to non-specific binding.
When planning your experiment, first determine the number of immunoprecipitation reactions
you plan to perform. Enough reagents are supplied in this kit to perform 20 total IP reactions:sufficient Ago1/2/3 Antibody is included to perform 10 miRNA Target IP reactions while
Negative Control IgG is included for 10 negative control IP reactions.
We recommended using 10 million cells per IP, and setting up reactions in duplicate. Pleaserefer to the descriptions included in the Buffer Preparation section for complete details on
buffer preparations, and to the Quick Chart for Preparing Buffers to determine the necessary
volumes based on the number of IP reactions.
Performing IP experiments in duplicate using both the Ago1/2/3 Antibody and the Negative
Control IgG will require 40 million cells that have been transfected with a miRNA of interest
and 40 million cells that have been transfected with a non-targeting control miRNA.
Sample TypemiRNA
Target IP

Negative
IgG IP

Total

Cells transfected with miRNA mimic

2 x 107 cells

2 x 107 cells

4 x 107 million

Cells transfected with non-targeting miRNA2 x 107 cells

2 x 107 cells

4 x 107 million

4

4

8

Number of IP reactions

Please see page 2 for a flow chart of the process.

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6

Growth of Cells
Prior to performing the immunoprecipitation reactions using cell lysates, growing cells willbe transfected with either a miRNA of interest or with a non-targeting control miRNA. We
recommended that you seed your cells to yield 70-80% confluence at the time you perform
your transfections, though the optimal amount may vary by cell line. Following transfection,
the cells will then be incubated for 8-24 hours before beginning the IP process by preparing
cell lysates.
The table below provides information for some commonly used cell culture dishes so you
can estimate how many you will need for your experiment. Keep in mind that the cells willcontinue to grow after the miRNA transfection. The number of cells present when you harvest
them to prepare lysates will depend upon a variety of factors including the cell type, theirhealth, the amount of time that elapses following the miRNA transfection and any effects thatthe miRNA may have upon cell growth.
As we recommend using 10 million cells per IP, and setting up reactions in duplicate, you willneed 20 million cells for each type of IP being performed. You may therefore need to grow,transfect and pool multiple dishes in order to have enough cells for your IP reactions.
Sample Type

6-well plate

60 mm dish10 cm dish15 cm dish

9

21

55

152

Seeding Density

0.3 x 106

0.8 x 1062.25 x 106

5.0 x 106

Cells at Confluency*

1.2 x 106

3.2 x 106

8.8 x 106

2.0 x 107

Growth Medium Volume

3 mL

5 mL

10 mL

20 mL

1X PBS (used per wash)

1 mL

2 mL

5 mL

10 mL

Complete Lysis Buffer(used per cell pellet)

100 µL100 µL150 µL150 µL

Surface Area (cm2)* The number of cells on a confluent plate or dish will vary with cell type. For this table, HeLa cells
were used. Please adjust the cell number as needed based on your particular cell type.

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7Buffer Preparation
Note:

RNA is highly susceptible to RNase degradation. Ensure all ancillary reagentsand tubes are RNase-free. Wear gloves at every step of the protocol (including
buffer preparation) to avoid RNase contamination from hands.

1X PBS
Prepare 31 mL of 1X PBS for every 15 cm dish for washing the cells prior to preparing thelysates. To a 50 mL conical tube add 27.9 mL sterile water and 3.1 mL 10X PBS. Mix byinverting. Place 1X PBS on ice to chill. Use the Quick Chart for Preparing Buffers to adjust thevolumes based on the number of dishes used. The volumes listed are based on the use of
one 15 cm dish per IP reaction. 1X PBS can be prepared in large quantities and stored at 4ºC
for 6 months.
1 M DTTDTT is provided as a 1 M stock solution. It is used as provided to prepare Complete Lysis
Buffer. In addition, a 100 mM DTT solution will be prepared in RNase-free water by adding
25 µL 1 M DTT to 225 µL RNase-free water. The 100 mM DTT solution is used to prepare the
Immunoprecipitation Buffer. Any unused 100 mM DTT solution should be stored at -20°C for
future use.
RNase Inhibitors
The RNase Inhibitors are supplied ready to use, and will be used to prepare Complete
Lysis Buffer.
Protease Inhibitor Cocktail (PIC)
Thaw the PIC at room temperature until fully dissolved, which takes about 30 minutes.
Vortex the PIC gently, spin down briefly, then add it to the Complete Lysis Buffer immediately
before use.
Ribonucleoside Vanadyl Complex
Ribonucleoside Vanadyl Complex is supplied ready to use. Thaw at room temperature andvortex briefly before use. It will be used to prepare Complete Lysis Buffer.
Complete Lysis Buffer
Complete Lysis Buffer should be prepared fresh before each experiment and discarded at
the end of the day. We recommend using a minimum of 150 µL Complete Lysis Buffer perpellet for cell numbers ranging from 5 - 20 million cells. For samples containing 1 - 5 million
cells, use 100 µL Complete Lysis Buffer per pellet. It is not advisable to use less than 100µL. See the Quick Chart for Preparing Buffers to prepare the amount of buffer needed foreach experiment.

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Protein G Magnetic Beads
Follow the instructions in Section C to conjugate the Ago1/2/3 antibody and the Negative
Control IgG to the beads for use in the IP reactions. For best results, gently shake and invert
the tube to resuspend the magnetic beads. The beads settle quickly, and therefore should beresuspended just before pipetting. We recommend cutting 2 mm from the end of a pipet tipprior to pipetting to prevent the tip from becoming clogged. The Protein G Magnetic Beads
are shipped on dry ice, and can be stored frozen until their first use. However, the beads
should not be re-frozen. After they have been thawed the Protein G Magnetic Beads should
be stored at 4ºC.
10X Wash Buffer AM7
Prepare a 1X Wash Buffer AM7 solution in RNase-free water. Use the Quick Chart for
Preparing Buffers to determine the amount of Wash Buffer needed for each experiment.
BSA Solution (50 mg/mL)
BSA is now provided ready to use in Wash Buffer AM7. Use 200 µL of the BSA Solution (50mg/mL) per 50 µL Protein G Magnetic Beads. Any unused solution should be dispensed into
aliquots and stored at -20°C for future use.
Ago1/2/3 Antibody
The supplied Ago1/2/3 antibody is a pan-Ago mouse monoclonal antibody that recognizes
Argonaute proteins Ago 1, Ago 2 and Ago 3. Use 5 µL (5 µg) antibody per IP reaction.
Negative Control IgG
The Negative Control IgG is used to determine the background in the immunoprecipitation
reactions. Use 25 µL (5 µg) Negative Control IgG per IP reaction.
0.5 M EDTA
The 0.5 M EDTA is supplied ready to use. It is used to prepare the
Immunoprecipitation Buffer.
10% SDS
The 10% SDS us supplied ready to use. However, SDS may precipitate upon storage. If
needed, warm the bottle to 37°C for 5 minutes and vortex to dissolve any precipitates. It is
used in Section E during the Proteinase K digestion.
Proteinase K (10 mg/mL)
The Proteinase K is supplied ready to use. It will be used to make fresh Proteinase K
Digestion Buffer in Section E.
Precipitation Buffer
The Precipitation Buffer is supplied ready to use. It is used in Section E to precipitate theRNA. Take care to not confuse this buffer with Immunoprecipitation Buffer, which you will
prepare fresh by following the Quick Chart for Preparing Buffers for use in Section D.

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Salt Solution
The Salt Solution is supplied ready to use. It is used in Section E to precipitate the RNA.
Glycogen
The Glycogen provided is at a concentration of 20 mg/mL. Make a 4-fold dilution of the
Glycogen stock by adding 120 µL RNase-free water directly to the provided tube to makea 5 mg/mL solution. Mark the tube as 5 mg/mL for future use. Use 5 µL of the diluted stock
per reaction.

Quick Chart for Preparing Buffers
Reagents to prepare

Components

1 IP

2 IPs

10 IPs20 IPs1X PBS

10X PBS

3.1 mL

6.5 mL

32 mL

65 mL

27.9 mL

58.5 mL

288 mL585 mLTOTAL REQUIRED

31 mL

65 mL

320 mL

650 mL1 M DTT

1.5 µL

3.5 µL

17 µL

33 µL

RNase Inhibitors

0.4 µL0.9 µL4.3 µL

8.3 µL

6 µL

14 µL

68 µL

132 µL

Ribonucleoside Vanadyl Complex

0.3 µL0.7 µL

3.4 µL6.6 µL

Lysis Buffer AM6

142 µL331 µL

1.61 mL3.12 mL

TOTAL REQUIRED

150 µL350 µL

1.7 mL

3.3 mL

RNase-free water

6.7 mL

18 mL

81 mL

144 mL

10X Wash Buffer AM7

740 µL

2 mL

9 mL

16 mL

TOTAL REQUIRED

7.4 mL

20 mL

90 mL

160 mL1X Wash Buffer AM7

850 µL1.89 mL

9.43 mL

18.9 mL

RNase Inhibitors

5 µL

11 µL

56 µL

111 µL

Ribonucleoside Vanadyl Complex

2 µL

4.5 µL

22 µL

45 µL

100 mM DTT

10 µL

22 µL

111 µL222 µL

0.5 M EDTA

34 µL

75 µL

378 µL756 µL

900 µL

2 mL

10 mL

20 mL

Sterile waterComplete Lysis Buffer

Protease Inhibitor Cocktail

1X Wash Buffer AM7

Immunoprecipitation
BufferTOTAL REQUIRED

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10

Protocols
Section A: miRNA Transfection
The protocol below is for transfection of miRNAs into HT1080 cells growing in 10 cm dishes
using DharmaFECT 4 Transfection Reagent, and is meant as a general guideline. You canscale the protocol as needed if you wish to grow your cells and transfect them in different
sizes of plates.
Moreover, the optimal transfection reagent and conditions will vary depending on what cells
are being transfected, what type of molecule is being transfected and what type of plate thecells are growing in. Dharmacon offers a Cell Type Guide on its website that details which of
its reagents have performed best for miRNA transfections into a variety of commonly usedcell lines. Please see http://dharmacon.gelifesciences.com/rnai-and-custom-rna-synthesis/
transfection/sirna-and-microrna-transfection for more information.
Be sure to set up enough plates so that you can perform transfection of your miRNA ofinterest as well as a non-targeting control miRNA, and so that there will be sufficient lysate
from each of the transfections to perform IP in duplicate using both the Ago1/2/3 Antibody
and the Negative Control IgG.
On Day 1, you will seed your cells in plates so that they grow to a confluency of 70-80% thefollowing day. You will then perform miRNA transfections on the plates and wait for 8-24
hours before preparing cell lysates. So, try to time when you seed your cells so that they will
be ready for transfection and lysate preparation at the desired timepoints.
1.

Trypsinize and count your cells, then seed 10 cm dishes with 2.25 x 106 cells in a
final volume of 15 mL antibiotic-free complete media per plate, which is a dilution of
150,000 cells/mL. (Complete media is the “standard” media your cells are maintainedin; it may contain serum.)2.

Grow cells overnight at 37°C with 5% CO2.

On Day 2, you will perform miRNA transfections on your cells after they have reached aconfluency of 70-80%. You will make up dilutions of each miRNA and the transfection reagent
in separate tubes using serum-free media, incubate, combine each diluted miRNA with thediluted transfection reagent and incubate again, add complete media to each mix, thenremove the media from the cells and add the appropriate transfection medium to each plate.
3.

For each miRNA transfection, prepare Mix 1 by adding 150 µL of miRNA (5 µM) to a
microcentrifuge tube followed by 1,850 µL of serum-free media.

4.

At the same time, prepare Mix 2 by adding 35 µL of DharmaFECT 4 to a
microcentrifuge tube followed by 1,965 µL of serum-free media.

5.

Gently mix the contents of each tube by pipetting up and down gently, then incubate
the mixes at room temperature for 5 minutes.

6.

Add the contents of Mix 2 to a 15 mL conical tube, then add Mix 1 to Mix 2 in thesame tube. Mix gently and incubate at room temperature for 20 minutes.

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11

7.

Add 11 mL of antibiotic-free complete media to the mixture made in step 6, thenmix gently.

8.

Remove the media from the dish of cells, then add the 15 mL of transfection mix tothe dish.

9.

Grow the cells at 37°C with 5% CO2 for 8-24 hours before preparing the cell lysate.

Section B: Lysate Preparation
This protocol describes cell lysate preparation from one 15 cm dish (approximately 1.5
x 107 cells). If you have grown your cells and transfected the miRNAs in plates that aresmaller than 15 cm, you can pool multiple plates of cells when the cells are scraped
from the plates (Step 3 below) to ensure that you have at least 1.0 x 107 cells for each
immunoprecipitation reaction.
1.

When cells are ready to harvest, prepare fresh Complete Lysis Buffer for each cellpellet. The volumes listed below are sufficient to process one 15 cm plate.

2.

Wash the plate twice with 10 mL ice-cold 1X PBS. (5 mL is sufficient for 10 cm plates.)3.

Add 10 mL ice-cold 1X PBS to each plate. Hold the plate at an angle and use a rubberpoliceman to scrape the cells down to collect them at the bottom edge of the plate.
Use a pipette to transfer the cells to a 50 mL conical tube on ice.

4.

Pellet the cells by centrifugation for 5 minutes at 1,500 rpm (453 x g) at 4°C.

5.

Remove the supernatant and discard. Resuspend each pellet in 1 mL ice-cold 1X PBS
by pipetting up and down. Keep samples on ice for the remainder of the procedure.

6.

Take a 10 µL aliquot of each cell pellet and determine the cell number usinga hemocytometer. It is important to use equivalent cell numbers for each
immunoprecipitation reaction.

7.

Transfer 10 million cells (1.0 x 107) of each sample to an RNase-free microcentrifuge
tube. We recommend setting up each IP reaction in duplicate.

8.

Centrifuge for 5 minutes at 1,500 rpm (453 x g) at 4°C. Remove the supernatant anddiscard. Resuspend each pellet in 150 µL ice-cold Complete Lysis Buffer by pipetting
up and down. (If using a different volume of cells, please refer to the chart on page 7.)

9.

Incubate lysate on ice for 5 minutes, then place tubes at -80°C. Keep the lysate
at -80°C for at least 1 hour while preparing the Protein G Magnetic Beads, as thisfreezing step helps to improve cell lysis. If desired, the lysate may be stored at -80°C
for up to 3 months.

Section C. Antibody Conjugation to Protein G Beads
The Protein G Magnetic Beads require blocking and washing prior to antibody conjugation.
To begin, determine the total number of immunoprecipitation reactions to be performed,
including both Argonaute (Ago) and negative IgG reactions, then label a separate RNase-freemicrocentrifuge tube for each.
For information regarding the use of the magnetic beads and the included bar magnet andglue dots, please refer to Appendix Section J.

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12Note:RNA is highly susceptible to RNase degradation. Ensure all ancillary reagentsand tubes used throughout the remainder of the protocol are RNase-free.
Wear gloves at every step of the protocol (including buffer preparation) to
avoid RNase contamination from hands.

1.

Resuspend the Protein G Magnetic Beads by end-over-end rotation of the vial for
5 minutes at room temperature. As magnetic beads have a tendency to settle tothe bottom of the tube, it is important to resuspend the contents completely before
use. Following resuspension, use a wrist flick to collect beads out of the cap prior to
opening the vial.

2.

Add 50 µL Protein G Magnetic Beads to each labeled tube.

3.

Add 200 µL BSA Solution (50 mg/mL) to each tube. Vortex 5 seconds. Allow tubes to
incubate with end-over-end rotation for 10 minutes at room temperature.

4.

Quick spin the tubes to collect the contents to the bottom. Place the tubes on themagnet to pellet the beads. Remove and discard the supernatant.

5.

Add 500 µL 1X Wash Buffer AM7 to each tube. Vortex 5 seconds. Place the tubes on
the magnet to pellet the beads. Remove and discard the supernatant.

6.

Wash a second time with 500 µL 1X Wash Buffer AM7. Vortex 5 seconds. Place thetubes on the magnet to pellet the beads. Remove and discard the supernatant, thenremove the tubes from the magnet.

7.

Add 100 µL 1X Wash Buffer AM7 to each tube and 5 µg of the appropriate antibody toeach of the labeled tubes.
Ago1/2/3 antibody:
Negative Control IgG:

Use 5 µL antibody per Argonaute IP reaction
Use 25 µL antibody per negative control IP reaction

8.

Incubate with end-over-end rotation for 30 minutes at room temperature.

9.

Centrifuge the tubes for 20 seconds at 9,000 rpm (8,150 x g) at room temperature tocollect the contents at the bottom of each tube.

10. Place the tubes on the magnet to pellet the beads. Remove and discard thesupernatant. Add 500 µL 1X Wash Buffer AM7 to each tube and vortex 5 seconds
to mix.
11. Repeat step 10 two additional times. Leaving the beads in the final wash solution,
place the tubes on ice.

Section D. RNA Complex Immunoprecipitation
In this section the antibody-conjugated beads will be combined with the lysate to
immunoprecipitate miRNA/mRNA complexes associated with Argonaute proteins (Ago1, Ago2
or Ago3). Background levels can be determined using Negative IgG-conjugated beads.
1.

Remove the lysate prepared in Section B from the freezer and thaw on ice.

2.

Freshly prepare Immunoprecipitation Buffer as described in the Buffer Preparation
section of the manual. Place the buffer on ice.

3.

Centrifuge the thawed lysate for 10 minutes at 14,000 rpm (20,817 x g) at 4°C.

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4.

During the lysate centrifugation, place the tubes of antibody-conjugated Protein G
Magnetic Beads from Section C, Step 11 on the magnet to pellet the beads. Remove
and discard the supernatant.

5.

Remove the tubes from the magnet and add 900 µL of Immunoprecipitation Buffer toeach tube of beads. Vortex 5 seconds to mix.

6.

Following lysate centrifugation, transfer 10 µL of each supernatant to a new RNasefree tube and label as Input. Store the Input samples at -80°C for use in Section E.

7.

Transfer the remaining supernatant (~100 µL) to the appropriate tube of antibodyconjugated beads. Discard the pellet.

8.

Incubate the IP reactions for 4 hours to overnight with end-over-end rotation at 4°C.

9.

Centrifuge the tubes for 20 seconds at 9,000 rpm (8,150 x g) at room temperature tocollect the contents at the bottom of each tube.

10. Place the tubes on the magnet to pellet the beads. Remove and discard thesupernatant. Add 500 µL ice-cold 1X Wash Buffer AM7 to each tube and vortex 5
seconds to mix.
11. Repeat step 10 five additional times for a total of 6 washes.
12. Place the tubes on the magnet to pellet the beads. Remove and discard thesupernatant. Add 10 µL 1X Wash Buffer AM7 to each tube to resuspend the beads.
Place the tubes on ice.

Section E. Proteinase K Digestion and RNA Purification
In this section the samples will be treated with Proteinase K and SDS to degrade the
Argonaute proteins, disrupt the antibody binding, and elute the RNA from the beads.
1.

Freshly prepare Proteinase K Digestion Buffer. Calculate the amount of Buffer needed
to process both the IP reactions and the Input samples.

Note:

It is important to add the chemicals in the order listed below to prevent
denaturation of Proteinase K by the concentrated SDS solution.
Reagent

1 reaction

No. of rxns

Volume Needed

1X Wash Buffer AM7

107 µL x___________=___________

10% SDS

15 µL x___________=___________

Proteinase K (10 mg/mL)

18 µL x___________=___________

140 µL x___________=___________

Total Volume

2.

Preheat a thermo-mixer or heat block to 55°C.

3.

Thaw the Input samples from Section D, Step 6 on ice.

4.

Add 140 µL of the freshly prepared Proteinase K Digestion Buffer to all tubes. This
includes the Input samples and the IP reactions from Section D, Step 12.

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5.

Incubate the samples at 55°C for 30 minutes with shaking. If a thermo-mixer is notaccessible, flick each tube by hand every 5 minutes and vortex once halfway throughthe incubation to resuspend the magnetic beads and ensure complete digestion.

6.

Centrifuge the tubes for 20 seconds at 9,000 rpm (8,150 x g) at room temperature tocollect the contents at the bottom of each tube.

7.

Place the tubes on the magnet to pellet the beads, then transfer the eluate to a new
RNase-free tube.

8.

Add 250 µL 1X Wash Buffer AM7 to each tube.

9.

Add 400 µL phenol:chloroform:isoamyl alcohol (25:24:1) to each tube.

Note:

We highly recommend purchasing phenol:chloroform:isoamyl alcohol rather
than mixing it yourself. This mixture can be difficult to homogenize, and if it is
not made properly, assay performance can suffer.

10. Vortex for 20 seconds, then centrifuge for 10 minutes at 14,000 rpm (19,722 x g) at
room temperature.
11. Transfer the aqueous (top) phase into a new RNase-free tube. Try to ensure
consistency in recovery for all samples. Add 400 µL chloroform to each tube.
12. Vortex for 20 seconds, then centrifuge for 10 minutes at 14,000 rpm (19,722 x g) at
room temperature.
13. Transfer the aqueous (top) phase into a new RNase-free tube. The volume will beapproximately 350 µL. Try to ensure consistency in recovery for all samples.
14. To prepare the samples for precipitation, add the following to each tube:
Reagent

Note:

1 reaction

No. of rxns

Volume Needed

Precipitation Buffer

50 µL x___________=___________

Salt Solution

15 µL x___________=___________

Glycogen (5 mg/mL)

5 µL x___________=___________

Absolute ethanol

875 µL x___________=___________

Total Volume

945 µL x___________=___________

If you collect more than 350 µL of the aqueous phase, or if you notice thatyour samples are freezing while stored at -80°C, add 900 µL of 100% ethanol
instead of 875 µL. If the samples freeze, poor signal will likely be seen.

15. Vortex the tubes for 20 seconds. Place tubes at -80°C for 1 hour to 24 hours toprecipitate the RNA.
16. Centrifuge for 30 minutes at 14,000 rpm (20,817 x g) at 4°C. Carefully discard thesupernatant without disturbing the pellet. If the pellet is not visible, leave ~100 µL of
the supernatant in the tube.
17. Add 500 µL of 80% ethanol to each tube. Centrifuge for 15 minutes at 14,000 rpm(20,817 x g) at 4°C. Carefully discard the supernatant without disturbing the pellet.

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Note:

If you do not plan to convert the RNA to cDNA immediately, the pellets may
be stored in 100 µL absolute ethanol for 2 weeks at -80°C. When ready touse, centrifuge for 15 minutes at 14,000 rpm (20,817 x g) at 4°C, then carefully
discard the supernatant and proceed with step 18.
Input samples will be used only for troubleshooting to verify that an mRNAtranscript was present in the original lysate prior to IP. Therefore, they caneither be converted to cDNA along with the other samples or stored asRNA. Because they will be more stable and you will be able to store themfor more than 2 weeks, we recommend to convert them to cDNA with theother samples.

18. Allow the pellets to air dry for 10 minutes at room temperature.
19. Resuspend pellets in 20 µL RNase-free water and place on ice. Proceed immediately
with Section F. Analysis of Samples by RT-PCR.

Section F. Analysis of Samples by qRT-PCR
The precipitated RNA can be analyzed by Reverse Transcription (RT) followed by real-time
PCR. We recommend using a 2-step RT-PCR system to first convert the RNA to cDNA. This
cDNA is then analyzed by qPCR. Follow the instructions that came with your specific RT-PCR
Kit to prepare your samples.
You will need to have already generated gene-specific qPCR primer set(s) that were
designed to amplify the cDNA that will be made from the mRNA transcript(s) you believe will
be bound by, and thus immunoprecipitated with, the miRNA of interest that was transfected
into your cells.
The IGF2 Primer pair included in this kit can be used in qRT-PCR as a positive control
to validate that your IP was successful. It shows strong enrichment following IP with ourpan-Ago antibody.
Please consult Appendix Section H for information on designing qPCR primers as well as
validating their function by determining their Primer Efficiency through the generation of astandard curve. Use only those primer pairs with an Primer Efficiency between 80-110%.
The following is an example preparation of cDNA using Applied Biosystems High-Capacity
RNA-to-cDNA Kit (Applied Biosystems, Catalog No. 4387406). Follow the instructions specificfor your Reverse Transcription Kit of choice.
1.

In a microcentrifuge tube on ice, prepare the RT reactions as follows.

2.

Vortex the samples for 5 seconds, then centrifuge for 1 minute at 1,500 rpm (453 x g)
at 4°C.

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Reagent

Sample / Input

No RT Control

2X RT Buffer

10 µL

10 µL

RNA (Sample or Input)

9 µL

9 µL

–

1 µL

1 µL

–

20 µL

20 µL

RNase-free water
Reverse Transcriptase
Total Volume

3.

To generate cDNA, place tubes in a thermal cycler at 37°C for 60 minutes, followedby 95°C for 5 minutes, then hold the samples at 4°C until you are ready to
perform qPCR.

4.

Prepare the primers for qPCR analysis. See the Recommendations for Primer Designin the Appendix. For the IGF2 control primer pair, use 1 µL of provided mix per PCR.

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Below is an example qPCR reaction. Please follow the instructions for your specific qPCR
instrument. We recommend using a commercially available SYBR Green qPCR master mix(e.g. Bio-Rad Cat # 170-8882) and preparing triplicate reactions.
5.

Prepare a dilution of cDNA for use in qPCR. The dilution factor required may varydepending on the abundance of the transcript of interest. We suggest a 2- to 5-folddilution of cDNA in RNase-free water as a starting point.

6.

In a microcentrifuge tube on ice, prepare a qPCR mix as follows. If you are preparing
a master mix, do not add the cDNA. Add each cDNA sample directly to the wells of
the plate or strip.
Reagent

Sample / Input

2X SYBR Green master mix

5 µL

Primer mix (2.5 µM each primer)

1 µL

RNase-free water

2 µL

cDNA (from Step 5 above)

2 µL

Total volume

10 µL

7.

Pipette the reactions into the wells of a reaction plate or strip appropriate for yourreal-time PCR system. Seal with optical adhesive film or optical caps, vortex thencentrifuge the reaction plate briefly. Use the run specifications dictated by yourmachine and reverse transcription kit. The example below applies when using theApplied Biosystems RNA-to-cDNA Kit and the Bio-Rad SYBR Green qPCR master mix
and qPCR machine.

8.

Place tubes in a real-time PCR instrument and program as below:
95°C for 2 minutes(95°C for 15 seconds, 58°C for 20 seconds, 72°C for 20 seconds) for 40 cycles
65°C for 5 seconds

9.

Analyze the results. Suggestions for data analysis can be found in Appendix Section Iof the manual.

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References
1.

Bomsztyk, K., et al. (2019) Nucleic Acids Research 47(12): e69.

2.

Kanter, J.E., et al. (2019) J. Clin. Invest. 129(10):4165-4179

3.

Levy, S., et al. (2022) Cell Reports 38, 110457

4.

Chapin, N., et al. (2022) Communications Biology 5:596

5.

Hopf, A., et al. (2022) Bioengineering (Basel) 9(9):412

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Appendix
Section G. Troubleshooting Guide
Problem/question

Recommendation

What is the fewest number ofcells that can be used for IP?

We recommend using 10 million cells in order to obtain the most consistent
results; the enclosed protocols were optimized using that amount of cells.
However, we have had success using 5 million cells, and have even beenable to go down to as few as 1 million cells in some instances. Your results
will vary depending on the cell type and how the cells respond to the miRNAtransfection.

Can I use any cell type? Whatabout cells from different
species?

You may use any cell type derived from humans or mice. The kit was
validated using 293, MCF-7, HT1080 and HeLa cells from humans, as wellas with 3T3 mouse cells. If you wish to work with cell types other than thosejust listed, it is suggested to use 10 million cells to ensure good enrichment.
Then, if the fold enrichment values are high, you can use 5 million cells for
subsequent reactions. This assay was not tested in any species other thanhuman and mouse, but it may work in other species. It is up to the discretionof the researcher as to whether the miRNA Target IP Kit is appropriate for
their cell type.

Can I use an antibody otherthan the supplied Ago 1/2/3
antibody?

Yes. If you have antibodies that have been validated to successfully
immunoprecipitate other RNA-binding proteins, then this kit should function
for use in RIP (RNA-binding Protein Immunoprecipitation). However, Active
Motif has not tested other antibodies, so results with other antibodies are notguaranteed.

Can I use theimmunoprecipitated/purified
mRNA molecules withconventional expression
microarrays or RNA-Seq?

Yes, the mRNA produced should be appropriate for these uses. However,
Active Motif has not tested these other applications, so cannot guarantee thatthey will work. Please consult the manufacturer's protocols of these alternate
methods.

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Problem/question

Recommendation

I don’t see any pellets after
the RNA Precipitation step.
Why?

It is important to use enough cells and/or antibody (preferably 5 million
and 5 µg) per reaction. It is crucial to include all of the listed components
when preparing buffers, such as RNase Inhibitors. It is necessary to havefrequent/continual mixing of the tubes during the proteinase K digestion step.
Collection of enough of the aqueous layer is necessary when performing thephenol:chloroform and chloroform extractions. Also, ensure that the mixture
does not freeze during RNA precipitation. If any of these guidelines are
not followed, you may not see a pellet. If all were followed, it is likely that
RNases were somehow introduced into your sample at some point during
the procedure. Ensure all ancillary reagents and tubes are RNase-free. Weargloves at every step of the protocol (including during buffer preparation) to
avoid RNase contamination from hands.

I saw a pellet after the RNA
precipitation step, but I’m notseeing the fold enrichment
I expected in my qRT-PCR.
Why?

It is possible that RNases could have degraded the RNA after step E-16,
before it was converted to cDNA. But, if you completed the RT reaction fairly
quickly after resuspending the pellets in water, this is unlikely. Ensure thatyour qPCR primer sets have an efficiency of 80-110%. Run your qRT-PCR
again. If you still do not get the results you expect, run qRT-PCR on the
Input samples to determine if the mRNA was present in the lysate prior toperforming IP. If it is, then either your RNA degraded or else the biology ofyour system may work differently than you thought. That is, the miRNA ofinterest may not bind to the mRNA transcripts you expected.

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21Section H: Recommendations for Primer Design
The following are a list of recommendations to consider when designing qPCR primers. We
recommend designing primers with the same annealing temperature to enable the use ofdifferent primer sets within the same experiment.
•

Design and analyze your potential primer pairs using an in silico PCR
program (i.e. Primer3 at http://frodo.wi.mit.edu/ or the UCSC Genome Browser
at http://genome.ucsc.edu/cgi-bin/hgPcr).

•

Primers that dimerize should be avoided as they will be bound by
SYBR Green, which will compromise accurate quantitation. You cantest your primers for self-complementarity and secondary structure
at http://frodo.wi.mit.edu/cgi-bin/primer3/primer3_www.cgi.

•

Ideally, the amplicons should be 50-100 bp in length with a GC content of 30-80%.

•

Avoid runs of identical nucleotides. If repeats are present, there must be fewer than
4 consecutive G residues. Make sure that the last 5 nucleotides at the 3´ end containno more than 2 G and/or C residues.

•

Primers should be designed to anneal optimally between 58°C to 60°C with arecommended length of 18-22 bp.

•

Use only primer sets that have an Primer Efficiency between 80-110%.

Determination of Primer Efficiency
Your qPCR primer set will be used to produce a standard curve that will enable you todetermine if the Primer Efficiency of your primers is sufficient. The slope of the standard
curve will also be used when calculating the Fold Enrichment of the IP performed using the
Ago1/2/3 Antibody
relative to the IP performed using the Negative Control IgG
1.

Produce a standard curve by performing qPCR with your primer set on known
genomic or cDNA quantities in triplicate. Run five samples that are 10-fold dilutions,e.g. 0.005 ng, 0.05 ng, 0.5 ng, 5 ng and 50 ng.

2.

The efficiency of the primers can be calculated using the slope of the standard curve
and the formula below. Many qPCR instrument programs will automatically calculate
the average of each of the triplicate reactions as well, as the Primer Efficiency.
Primer Efficiency = [10(-1/slope) – 1] x 100%
For example, if the slope = -3.508
Primer Efficiency = [10(-1/slope) – 1] x 100% = [10(-1/-3.508) – 1] x 100%
Primer Efficiency = [1.928 - 1] x 100% = 0.928 x 100%
Primer Efficiency = 92.8%
The ideal efficiency is 100% ±10% (i.e. 90-110%, where -3.32 = 100% efficiency).

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22Section I: Data Analysis
Below is a strategy that can be used to determine the Fold Enrichment of each Argonaute IP
reaction as compared to its equivalent Negative Control IgG IP reaction. You will need touse the slope of the standard curve that was generated when you determined the Primer
Efficiency for each qPCR primer set.
Calculate Fold Enrichment
1.

Average the triplicate CT values for each Argonaute and Negative Control IgG
reaction. Some machines may average your triplicate reactions automatically.

2.

Using the slope of the standard curve that was already determined in Section H andthe formula below, calculate the Fold Enrichment of each Argonaute IP in relation to
the corresponding Negative control IP.
Fold Enrichment = AE(Neg IgG CT – Ago CT)
where AE = Amplification Efficiency = 10(-1/slope)For example, if the slope = -3.508, the Neg IgG CT = 32.04, and the Ago CT = 29.19
Amplification Efficiency = 10(-1/slope) = 10(-1/-3.508) = 1.928
Fold Enrichment = 1.928(32.04 – 29.19) = 1.928(2.85) = 6.5

Section J. Use of Magnetic Beads and Included Bar Magnet
Caution: The included neodymium bar magnet is extremely powerful and is easily
broken if handled incorrectly.
•• The magnet should be stored in the provided tube.

•• Be careful when working near metal objects or surfaces. A free magnet will jump
great distances onto nearby metal surfaces with surprising speed. This can breakthe magnet.

•• Use the provided Mini Glue Dots to attach the bar magnet to an empty pipette
tip box to create an effective magnetic stand for use with either PCR strips or
microcentrifuge tubes.

•• If the magnet becomes attached to a flat metal surface, it should be removed bysliding it off the edge of the surface. The magnet may break if you attempt to pull one
end or pry it away from the metal.

Creating a magnetic stand for 8-well PCR strips:
Note:
1.

8-well strip tubes for use with standard 96-well PCR cyclers are recommended(e.g. Thermo Fisher AB-0451).

Place a strip of PCR tubes in the wells of an empty tip box (200 µL tips) and place themagnet directly against the tubes. This is the way the magnet will be positioned when
the glue dots are used to affix it to the box.

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2.

Remove the covering tape from one side of two glue dots and attach the glue dotson the bar magnet (the uncovered face of the dot is placed on the magnet) as
shown below.

3.

Remove the covering tape from the exposed side of the glue dots. Fix the magnet
to the tip box so that it is against the PCR tubes. The magnetic stand is now readyfor use.

Note:

Familiarize yourself with using the magnetic stand before performing with PCR
tubes for the first time. Add 5 µL of magnetic beads to 100 µL Wash Buffer in
one tube of an 8-well strip of PCR tubes. Use this tube with the assembled bar
magnet stand to become familiar with use of the beads and magnet. It
is difficult to re-suspend the beads if the tubes are directly adjacent to
the magnet, so it is best to move the tubes away from the magnet forresuspension steps.

Washing should be performed as follows:
1.

Place the tubes in the rack against the magnet and allow the beads to be pinned tothe side of the tube, as shown below.

2.

Remove supernatant with a 200 µL pipetteman or a 200 µL eight-channel pipetteman.

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3.

Move the tube strip into a row that is not adjacent to the magnet.

4.

Add wash buffer and pipet up and down to fully re-suspend the beads. Ensure that aminimal amount of beads cling to the tips when the re-suspension is complete.

5.

Repeat steps 1-4 until desired washing steps are complete.

Centrifugation of 8-well PCR strip tubes:
When working with 8-well PCR strip tubes, it may be desirable to centrifuge the tubes tocollect the liquid and beads from the insides of the caps. This is easily accomplished in acentrifuge fitted with adaptors for spinning microtiter plates. Place a standard 96-well platein the adaptor to hold the tubes in place. Be sure to balance the rotor (i.e. place a microtiterplate and tubes of appropriate mass in the rotor’s opposing 96-well plate adaptor). Spin theplates briefly to let the rotor reach a speed of 1000 x g before allowing the rotor to stop.
Creating a magnetic stand for microcentrifuge tubes:
1.

Remove the covering tape from one side of two glue dots.

2.

Place two microcentrifuge tubes in the wells of an empty tip box (1000 µL) and place
the magnet directly against the tubes. This is the way the magnet will be positioned
when the glue dots are used to affix it to the box.

3.

Attach the glue dots on the bar magnet (the uncovered face of the dot is placed on
the magnet) as shown above.

4.

Remove the covering tape from the exposed side of the glue dot. Fix the magnet to
the tip box so that it is against the tubes. The magnetic stand is now ready for use.

Note:

Microcentrifuge tubes are held less securely in this assembled tube standthan in a typical commercial magnetic stand. This is not a problem if the below
washing protocol is followed. Work with 1 tube at a time, and keep the tubes
in the standard tube rack unless you are holding the tube next to the magnet.

Washing is best performed one tube at a time, as follows:
1.

Place the tube in a standard microcentrifuge tube rack and open the cap.

2.

Place the opened tube in the assembled magnetic stand. The beads will pellet
more rapidly if the bottom of the tube is held against the magnet, as shown below,
and then slowly lowered into the well. This will pellet the beads up onto the side of
the tube.

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3.

Allow the beads to pellet completely and remove supernatant with a 1000 µL
pipetteman. You can either leave the tube in the rack or pull it out when you remove
the buffer. The beads will remain on the side of the tube, even when not next tothe magnet.

4.

Return the tube to the standard microcentrifuge tube rack, add 800 µL wash buffer
and fully resuspend the beads by pipetting up and down.

5.

Repeat steps 2-4 until desired washing steps are complete. After the final wash
has been removed, the last traces of wash buffer should be removed with a 200µL pipetteman.

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Technical Services

China

Japan

EuropeNorth America

If you need assistance at any time, please call or send an e-mail to Active Motif Technical
Service at one of the locations listed below.

Toll free:877 222 9543

Direct:

760 431 1263

Fax:

760 431 1351

E-mail:tech_service@activemotif.com

Direct:+32 (0)2 653 0001

Fax:

+32 (0)2 653 0050

E-mail:eurotech@activemotif.com

Direct:+81 (0)3 5225 3638

Fax:

+81 (0)3 5261 8733

E-mail:japantech@activemotif.com

Direct:(86)-21-20926090

Cell Phone:
E-mail:18521362870
techchina@activemotif.com

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