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Protocol: In Vitro Transcription (IVT) for mRNA Synthesis

 

Protocol: In Vitro Transcription (IVT) for mRNA Synthesis

1. Template Preparation (Linearization and PCR Amplification)

a. Linearization of Plasmid

- Digest 5–10 µg of pUC57AG-GOI (e.g., pUC57AG_Luc) using XbaI at 37 °C.
- Run the digested product on a 1% agarose gel (60 mL TAE buffer + 0.6 g agarose + 6 µL GelRed).
- Load: 5 µL DNA + 1 µL DNA loading dye; Run at 120 V for 30 min.
- Purify the linearized product using a commercial PCR Purification Kit.
- Quantify the DNA using Nanodrop, adjust to 1 ng/µL, and name it as L_pUC57AG_GOI.

b. PCR Amplification (to add polyA tail)

Use a KOD hot-start kit for PCR:

Component

Volume (µL)

10× KOD buffer

5

25 mM MgSO₄

3

2 mM dNTP mix

5

5′ Primer (10 µM)

1.5

3′ Primer (10 µM)

1.5

Template DNA (12.5 ng)

12.5

KOD Polymerase

1

Nuclease-free water

up to 50 µL (20.5 µL)

PCR conditions:
- 94 °C for 2 min
- [98 °C 10 sec → 62 °C 30 sec → 68 °C 1 min] × 30 cycles
- Final hold: 4 °C

- Purify PCR product using a PCR Purification Kit
- Quantify with Nanodrop and adjust to 100 ng/µL; label as GOI-polyA
- Check 1 µL product on 1% agarose gel (120 V, 40 min)

2. In Vitro Transcription (IVT)

Use Thermo MEGAscript T7 Kit for IVT reaction:

Component

Volume (µL)

10× T7 reaction buffer

2

ATP (A)

2

CTP (C)

2

GTP (G)

2

UTP (U)

2

Template (GOI-polyA)

0.5–1 µg (~7 µL if 700 ng)

T7 RNA Polymerase Mix

2

CleanCap AG (100 mM)

1

Incubate at 37 °C overnight.

3. Post-IVT Processing

a. DNA Removal
- Add 1 µL TURBO DNase
- Incubate at 37 °C for 15 min

b. RNA Purification
- Use MEGAclear Kit
- Quantify purified mRNA (expected: 100–200 µg)
- Store at –80 °C for long-term use

4. Quality Check

- Run purified mRNA on 1% agarose gel alongside mRNA ladder
- Use Nanodrop to check concentration and purity

Primers Used

5′ Primer (T7 promoter): TTGGACCCTCGTACAGAAGCTAATACG
3′ Primer: (120×T) ACTTCCTACTCAGGCTTTATTCAAAGACCA

Materials and Reagents

- Plasmid: pUC57AG-GOI
- Enzymes: XbaI (ER0685), TURBO DNase (AM2239)
- Kits: KOD Hot Start kit (71086-3), Thermo MEGAscript T7 (amb1334-5), MEGAclear (AM1908), PCR Purification Kit (28104)
- Primers (see above)
- Reagents: CleanCap AG (E2080), mRNA ladder (SM1821), DNA ladder (SM1334), DNA loading dyes (R1151)

 

By Xuehao Zhang

05/22/2025

MEGAclear™ Kit Procedure

CAUTION! Filter Cartridges should not be subjected to RCFs over 16,000 × g because it could cause mechanical damage and/or may deposit glass filter fiber in the final sample.

 

Before using the kit for the first time Prepare the Wash Solution

Add 20 mL of ACS grade 100% ethanol to the bottle labeled Wash Solution Concentrate. Mix well. Place a check in the box on the label to indicate that the ethanol was added. With ethanol, this solution will be referred to as Wash Solution.

 

1.       Bring the RNA sample to 100 μL with Elution Solution. Mix gently but thoroughly.

 

2.       Add 350 μL of Binding Solution Concentrate to the sample. Mix gently by pipetting.

 

 

3.       Add 250 μL of 100% ethanol to the sample. Mix gently by pipetting.

 

4. Apply the sample to the filter:

a. Insert a Filter Cartridge into 1 of the Collection and Elution Tubes supplied.

b. Pipet the RNA mixture onto the Filter Cartridge.

c. Centrifuge for ~15 sec to 1 min, or until the mixture has passed through the filter. Centrifuge at RCF 10,000–15,000 × g (typically 10,000–14,000 rpm). Spinning harder than this may damage the filters.

d. Discard the flow-through and reuse the Collection and Elution Tube for the washing steps.

 

5. Wash with 2 × 500 μL Wash Solution.

Note: Make sure that the ethanol has been added to the Wash Solution Concentrate before using it.

a. Apply 500 μL Wash Solution. Draw the Wash Solution through the filter as in the previous step.

b. Repeat with a second 500 μL aliquot of Wash Solution.

c. After discarding the Wash Solution, continue centrifugation or leave the Filter Cartridge on the vacuum manifold for 10–30 sec to remove the last traces of Wash Solution.

 

6. Elute RNA from the filter with 50 μL Elution Solution using one of the methods described below; they are equivalent in terms of RNA recovery.

• RNA elution

a. Place the Filter Cartridge into a new Collection/Elution Tube.

b. Apply 50 μL of Elution Solution to the center of the Filter Cartridge. Close the cap of the tube and incubate in a heat block set to 65–70°C for 5–10 min.

c. Recover eluted RNA by centrifuging for 1 min at RT (RCF 10,000–15,000 × g).

d. To maximize RNA recovery, repeat this elution procedure with a second

50 μL aliquot of Elution Solution. Collect the eluate into the same tube.

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