Dynabeads
™
-Based Solid-Phase In Vitro Transcription and RNA
Purification
Catalog Numbers
65005D, 65006D, 65020D, 65021D, 65040D, 65042D, 65030D, 65032D, 65034D, 65036D
Pub.No. MAN0029518 Rev. A.0
WARNING! Read the Safety Data Sheets (SDSs) and follow the handling instructions. Wear appropriate protective eyewear,
clothing, and gloves. Safety Data Sheets (SDSs) are available from thermofisher.com/support
.
■
Product description ........................................................................................ 1
■
Contents and storage ...................................................................................... 2
■
Required materials not supplied .............................................................................. 3
■
Before you begin .......................................................................................... 3
■
Part A: Protocol for RNA synthesis with Dynabeads
™
Streptavidin for In Vitro Transcription ................................ 4
■
Part B:
Workflow for RNA purification using Dynabeads
™
Carboxylic Acid for RNA Purification in combination with Dynabeads
™
RNA
Binding Buer ............................................................................................ 5
Product description
Part A:
Dynabeads
™
Streptavidin for In Vitro Transcription
contains monosized (1 µm) paramagnetic beads which are characterized by
high speed to magnet. The product concentration is 10 mg/mL, stored in PBS buer containing 0.01% Tween-20 and 20% ethanol is
added as preservative.
The biotinylated template is made by PCR amplification of the target sequence in a plasmid construct or in a synthetic DNA construct,
by using a biotinylated forward primer, located 30-100 base pairs upstream of the T7-promoter and a non-biotinylated reverse primer.
The biotinylated template can be immobilized directly to Dynabeads
™
Streptavidin for In Vitro Transcription without the need for prior
purification.
The bead-bound template is used directly in the in vitro transcription (IVT), then removed from the synthesized RNA
by magnetic separation and can be reused at least six times in consecutive IVT reactions (see Figure1). As an alternative to PCR-
amplification, a linearized plasmid can be biotinylated by a fill in reaction of biotin-dUTP in the 5-prime overhang sequence, provided the
correct plasmid design.
Part B:
Dynabeads
™
Carboxylic Acid for RNA
Purification
are monosized (1 µm) paramagnetic beads which are characterized by high
speed to magnet and high pellet stability. The product concentration is 10 mg/mL, stored in purified water. The product is used in
combination with Dynabeads
™
RNA Binding
Buer
for purifying the crude RNA produced by solid-phase IVT (see Figure1). The RNA
is mix
ed with the beads and the buer is added. The RNA is thereby bound to the beads surface and remaining components of the
reaction mixture are removed by applying a magnet and discarding the supernatant. The beads may be re-cycled at least six times. The
Dynabeads
™
RNA Binding Buer needs to be protected from light exposure during storage.
USER GUIDE
For Research Use Only. Not for use in diagnostic procedures.
Bead-Template Reuse
In Vitro Transcription
Immobilization
of Template to
Dynabeads™
Streptavidin for In
Vitro Transcription
Bead-Template
Removal by Magnet
Crude RNA
Purified RNA
Purification by
Generic Capture
Bead Reuse
Biotinylated Template
Synthetic DNA
PCR Amplification
Bead-Template Complex
Linearized pDNA
Biotin Fill-in
OR
Plasmid
Part A
Part B
RNA Purification
on Dynabeads™
Carboxylic Acid
for RNA Purification
with Dynabeads™
RNA Binding Buffer
Part A
Part B
Figure1 Work
flow for RNA synthesis with Dynabeads
™
Streptavidin for In Vitro Transcription (Part A) and RNA purification on
Dynabeads
™
Carboxylic Acid for RNA Purification (Part B). Beads in both parts may be re-cycled at least six times.
The following protocols (see Figure1) describe IVT of a biotinylated DNA template immobilized on Dynabeads
™
Streptavidin for In Vitro
Transcription (Part A), and purification of synthesized RNA on Dynabeads
™
Carboxylic Acid for RNA Purification in combination with
Dynabeads
™
RNA Binding Buer (Part B).
The products used here are intended for basic research applications and drug discovery screening. The protocols are highly scalable and
automation-ready with KingFisher
™
instruments.
To learn more about products and support documentation available for GMP-compliant manufacturing of RNA vaccines and
therapeutics visit: thermofisher.com/dynabeadsmrna.
Contents and storage
Product Cat. no. Amount
[1]
Storage
Part A
Dynabeads
™
Streptavidin for In Vitro Transcription
65005D 2 mL
Store at 2–8°C.
65005D 10 mL
2 Dynabeads
™
-Based Solid-Phase In Vitro Transcription and RNA Purification User Guide
Product Cat. no. Amount
[1]
Storage
Part B
Dynabeads
™
Carboxylic Acid for RNA Purification
65020D 2 mL
Store at 2–8°C.
65021D 10 mL
Dynabeads
™
RNA Binding Buer
65040D 20 mL
Store at 2–8°C. Protect from light exposure during storage.
65042D 50 mL
[1]
Concentration is 10 mg/mL
Required materials not supplied
Unless otherwise indicated, all materials are available through thermofisher.com. "MLS" indicates that the material is available from
fisherscientific.com or another major laboratory supplier.
Catalog numbers that appear as links open the web pages for those products.
Item
Source
Parts A and B
DynaMag
™
-2 Magnet thermofisher.com/magnets
Thermal mixer
[1]
13687717
UltraPure
™
DNase/RNase-Free Distilled Water 10977035
RNase-free tubes (e.g. RNase-free Microfuge Tubes, 1.5 mL) AM12400
Qubit
™
RNA BR Assay Kit Q10210
Part A
1 M Tris, pH 8 AM9855G
IVT kit of choice (e.g. Invitrogen
™
MEGAscript
™
T7 Transcription Kit) AMB13345
Platinum
™
SuperFi II PCR Master Mix 12368010
Part B
70% ethanol
[2]
MLS
TE-buer, pH 7, RNase-free AM9861
1 M Tris, pH 8, RNase-free AM9850G
[1]
Thermal mixer with tilting and/or rotation of tubes to keep beads in suspension during template immobilization and the IVT reaction.
[2]
Wash buffer
Before you begin
To produce the biotinylated template by PCR, it is recommended to use a high-fidelity system such as Platinum
™
SuperFi II PCR Master
Mix. The poly-A tail can either be introduced with the reverse primer (with poly-T tail) during PCR or can be part of the sequence.
Dynabeads
™
-based solid-phase IVT is compatible with reagents such as Invitrogen
™
MEGAscript
™
T7 Transcription Kit, Invitrogen
™
mMESSAGE mMACHINE
™
T7 Transcription Kit, and Invitrogen
™
mMESSAGE mMACHINE
™
T7 ULTRA Transcription Kit based on the user
needs and strategies for capping and polyadenylation.
Prepare a biotinylated PCR product including the T7-promoter upstream of the 5’ UTR (untranslated region) and ORF (Open Reading
Frame), optionally with a defined polyA-tail in the 3’ end. The forward primer must be biotinylated (HPLC purified) and have a distance
to the T7-promoter of at least 30–100bp. Dilute the PCR product to 20–40 ng/µL in 10mM Tris, pH 8 or nuclease-free water before
immobilization to the beads.
For plasmid as template, a linearized plasmid can be biotinylated by a fill in reaction of biotin-dUTP in the 5-prime overhang sequence,
provided the correct plasmid design.
For each 100 µL IVT reaction, prepare 100 µl (1 mg) beads and 50 µl of 20–40 ng/µl (1–2 µg) biotinylated template (final 1–2 µg
template/mg bead). This amount is a suggested starting point for a 2.5 kb template and may be optimized for dierent templates and
Dynabeads
™
-Based Solid-Phase In Vitro Transcription and RNA Purification User Guide 3
sizes. More than 2-3 mg beads may result in too high density of the template on the beads surface and reduce RNA yield. IVT reaction
volume can also be scaled up and down in a linear fashion, by adjusting all the components correspondingly.
Part A: Protocol for RNA synthesis with Dynabeads
™
Streptavidin for In Vitro Transcription
Buers needed
• 2X Streptavidin Binding and Washing Buer (10 mM Tris-HCL, pH 7.5, 1 mM EDTA, 2 M NaCl)
• 10 mM Tris pH 8
Prepare Dynabeads
™
Streptavidin for In Vitro Transcription
1. Resuspend the Dynabeads
™
Streptavidin for In Vitro Transcription completely by vortex mixing.
2. Place the vial with Dynabeads
™
magnetic beads on a roller for at least 20 minutes.
3. Transfer 100 µL (= 1 mg) resuspended Dynabeads
™
magnetic beads to an RNase-free tube.
4. Place RNase-free tube containing Dynabeads
™
magnetic beads on magnet for 15–30 seconds, then remove supernatant.
5. Wash Dynabeads
™
magnetic beads once in 100 µL 2x streptavidin binding and washing buer, by resuspending using a pipette or
brief vortex mixing.
6. Place Dynabeads
™
magnetic beads on magnet for 15 seconds, then remove the supernatant.
7. Resuspend Dynabeads
™
magnetic beads in 50 µL 2x streptavidin binding and washing buer.
Immobilize biotinylated template
1. Add 50 µL (1–2 µg) biotinylated PCR-product to the Dynabeads
™
magnetic beads suspension.
2. Rotate on thermal mixer at 1,500 RPM for 30 minutes at room temperature.
3. Place biotinylated PCR-product immobilized beads on magnet for 15 seconds, then remove supernatant.
4. Wash the DNA-bead complex by resuspending in 100 µL 10mM Tris pH 8, using a pipette or brief vortex mixing, then remove the
supernatant.
5. Repeat the wash of the DNA-bead complex three times for a total of four washing steps. For the last washing step, do not remove
the supernatant. Proceed to “In vitro transcription” on page5.
4 Dynabeads
™
-Based Solid-Phase In Vitro Transcription and RNA Purification User Guide
Prepare IVT reaction mix
MEGAscript
™
T7 Transcription Kit scaled up to 100 µL reaction volume is shown in table below.
IMPORTANT! Keep all the reagents on ice, except the 10x IVT reaction buer, while using the kit.
Reagents Kit manual: 1x for 20 µL reaction volume(µL)/reaction Scale up: 5x for 100 µL reaction volume (µL)/reaction
[1]
Nuclease-free water
[2]
8µL 40µL
ATP 2µL 10µL
CTP 2µL 10µL
GTP 2µL 10µL
UTP 2µL 10µL
10x reactions buer 2µL 10µL
Enzyme mix 2µL 10µL
Template Bead pellet
[3]
Bead pellet
Total volume 20 100
[1]
The reaction is directly scalable, by increasing or decreasing all components.
[2]
Included in kit
[3]
The volume of the bead pellet does not need to be subtracted.
In vitro transcription
1. Place DNA-bead complex from step5 on magnet, then discard supernatant.
2. Resuspend DNA-bead complex in 100µL
MEGAscript
™
IVT-reaction mix.
3. Incubate in thermal mixer at 1,500 RPM at 37°C for 1–3 hours.
Note
:
The reaction time in this step should be optimized for each template and desirable RNA yield.
4. Place tubes on magnet for 15–30 seconds, then transfer the supernatant containing the RNA to new, RNase-free tubes.
5. Place on ice prior to analysis or freeze at -70°C.
6. Measure RNA concentration.
Note
:
We recommend using the Qubit
™
RNA Broad Range (BR) Assay Kit.
Note: The RNA yield per milligram of beads will depend on several parameters such as template density immobilized on the bead
surface and length of the IVT reaction. A 100 µL 2-hour reaction typically yields 4 µg/µL RNA, giving up to 400 µg per reaction. The
RNA yield can vary between dierent biotinylated templates. The DNA-bead complex may be reused in successive IVT reactions at
least six times by adding new IVT r
eagent mix to the magnetic bead-DNA complex.
Part B: Workflow for RNA purification using Dynabeads
™
Carboxylic Acid for RNA Purification in
combination with Dynabeads
™
RNA Binding
Buer
Recommended starting volumes
The following protocol describes generic capture purification of RNA from the IVT reaction. This purification removes NTPs, proteins and
other remaining components of the IVT reaction mix.
We recommend using the same volumes of RNA Binding
Buer and the input RNA solution. This protocol is scalable. Users should keep
the ratio of volumes, RNA and beads constant, but the relative volumes of wash buer are more flexible.
Use this experimental set-up as a suggested starting point:
Dynabeads
™
-Based Solid-Phase In Vitro Transcription and RNA Purification User Guide 5
Reagent Volume
Crude IVT mix
[1]
100 µL
[2]
Dynabeads
™
Carboxylic Acid for RNA Purification 30 µL
[3]
Dynabeads
™
RNA Binding Buer 100 µL
Elution output 100 µL
[1]
Input RNA solution
[2]
For 2500 nt-long RNA, we have tested concentrations up to 5 µg/µL with over 90% recovery.
[3]
300 µg
Prepare RNA samples and beads
1. If necessary, adjust the concentration of RNA by diluting to 5 µg/µL or lower with 10 mM Tris-HCl, pH 7.0. Place on ice.
2. Vortex Dynabeads
™
Carboxylic Acid for RNA Purification for 10 seconds, then place on a roller for ≥ 20 minutes at room temperature.
3. For each sample to be purified, transfer 30 µL (300µg) of resuspended Dynabeads
™
Carboxylic Acid for RNA Purification to
RNase-free tubes.
4. Place the tubes on the magnet until the solution looks clear (approximately 30-seconds), then discard the supernatant.
5. Wash beads in 100µL UltraPure
™
DNase/RNase-Free Distilled Water.
6. Place tubes on magnet, then discard the supernatant. Proceed to “Bind RNA to beads” on page6.
Bind RNA to beads
1. For each sample to be purified, add 100 µL crude RNA dilution to the 300 µg bead pellet, then mix by pipetting until beads are well
resuspended.
2. Add 100 µL RNA Binding Buer, then mix gently by pipetting until homogenous suspension until the suspension becomes
endogenous..
3. Incubate in thermal mixer at 1,000 RPM at room temperature for 10 minutes.
4. Place the tubes on the magnet until the solutions look clear (approximately 1-2 minutes), then discard the supernatant.
Wash and dry RNA-bead complex
1. Add 500 µL of the Wash Buer (70% ethanol) to the RNA-bead complex, then resuspend the beads by vortexing.
Note: Some aggregation of the beads during the washing steps should be expected.
2. Place the tubes on the magnet until the solution looks clear (approximately 30-60 seconds), then discard the supernatant.
3. Repeat step1 and step2 twice for a total of three washes.
4. Place on magnet, then remove all residual Wash Buer including any Wash Buer in the lid.
5. Dry the bead pellet for 10 minutes at room temperature while on the magnet.
Note: During drying step, keep the tube lid open. Make sure that all residual Wash Buer is removed after 10 minutes of airdrying.
Elute RNA
1. Remove the tubes from the magnet, then add 100 μL of Elution Buer (TE buer, pH 7 or another buer of choice).
2. Re-suspend the beads by pipetting.
3. Incubate on thermal mixer at 1,000 RPM at room temperature for 5 minutes.
4. Place the tubes on magnet and transfer cleared supernatant to new RNase-free tubes.
6 Dynabeads
™
-Based Solid-Phase In Vitro Transcription and RNA Purification User Guide
5. Place the tubes on ice and proceed with analyses.
6. Measure RNA concentration.
Note: We recommend using the Qubit
™
RNA BR Assay Kit or NanoDrop
™
One Microvolume UV-Vis Spectrophotometer.
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Revision history:Pub.No.MAN0029518 A.0
Revision Date Description
A.0 10 July 2023
New document for Dynabeads
™
-Based Solid-Phase In Vitro Transcription and RNA Purification.
The information in this guide is subject to change without notice.
DISCLAIMER: TO THE EXTENT ALLOWED BY LAW, THERMO FISHER SCIENTIFIC INC. AND/OR ITS AFFILIATE(S) WILL NOT BE LIABLE FOR SPECIAL, INCIDENTAL, INDIRECT,
PUNITIVE, MULTIPLE, OR CONSEQUENTIAL DAMAGES IN CONNECTION WITH OR ARISING FROM THIS DOCUMENT, INCLUDING YOUR USE OF IT.
Important Licensing Information: These products may be covered by one or more Limited Use Label Licenses. By use of these products, you accept the terms and conditions of all
applicable Limited Use Label Licenses.
©2023 Thermo Fisher Scientific Inc. All rights reserved. All trademarks are the property of Thermo Fisher Scientific and its subsidiaries unless otherwise specified.
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10 July 2023