Nov 17, 2024

Public workspacemRNA Synthesis Protocol using the HiScribe T7 mRNA Kit with CleanCap Reagent AG

DOI
dx.doi.org/10.17504/protocols.io.e6nvw11d7lmk/v1
  • 1Washington University
Cecilia Escudero
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DOI: dx.doi.org/10.17504/protocols.io.e6nvw11d7lmk/v1
Protocol Citation: Carolina Lopez 2024. mRNA Synthesis Protocol using the HiScribe T7 mRNA Kit with CleanCap Reagent AG. protocols.io https://dx.doi.org/10.17504/protocols.io.e6nvw11d7lmk/v1
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License,  which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: May 30, 2024
Last Modified: November 17, 2024
Protocol Integer ID: 100962
Abstract
Protocol for mRNA preparation
Standard mRNA Synthesis Protocol using the HiScribe T7 mRNA Kit with CleanCap Reagent AG (NEB #E2080)
Standard mRNA Synthesis Protocol using the HiScribe T7 mRNA Kit with CleanCap Reagent AG (NEB #E2080)
2h 15m
2h 15m
BEFORE STARTING
  • Recommend wearing gloves and using nuclease-free tubes and reagents to avoid RNase contamination. Reactions are typically 20 µl but can be scaled as needed.
  • Thaw the necessary kit components to room temperature (Reaction Buffer, NTPs, CleanCap Reagent AG and Template DNA), mix and microfuge briefly to collect solutions to the bottom of the tubes. Keep the Enzyme Mix on ice.
  • If several reactions will be performed a master mix of Reaction Buffer, NTPs and CleanCap can be prepared according to the volumes per reaction below. Use 12 µl of the Master Mix per reaction.
1 - Set up the following reaction at room temperature in the following order:

2 - Gently mix the reaction by pipetting up and down and microfuge briefly. Incubate at Temperature37 °C for Duration02:00:00 .
For reaction times of 60 minutes or less, a water bath or heat block may be used. For reactions longer than 60 minutes, we recommend using a dry air incubator or PCR instrument set to Temperature37 °C (with a heated lid) to prevent evaporation. For reactions with transcripts <0.3 kb incubations up to 16 hours or overnight can be performed.

3 - Bring the reaction volume up to Amount50 µL with nuclease-free water. Add Amount2 µL of DNase I, mix well and incubate at Temperature37 °C for Duration00:15:00 .

4 - Proceed with mRNA purification.
2h 15m
Monarch RNA Cleanup Kit Protocol (after IVT) NEB #T2030
Monarch RNA Cleanup Kit Protocol (after IVT) NEB #T2030
3m
3m
BEFORE STARTING
  • The standard protocol outlined below will purify RNA ≥ 25 nt. A simple modification in Step 2 can allow for the purification of RNA as small as 15 nt.
  • Centrifugation should be carried out at 16,000 x g in a standard laboratory microcentrifuge at room temperature.
  • Add 4 volumes of ethanol (>95%) to one volume of RNA Cleanup Wash Buffer


  1. Add Amount100 µL Cleanup Binding Buffer to the Amount50 µL sample. A starting sample volume of Amount50 µL is recommended. For smaller samples, nuclease-free water can be used to adjust the volume. For samples larger than 50 μL, scale buffer volumes accordingly. Samples with a starting volume > 150 μL will require reloading of the column during Step 3.
  2. Add Amount150 µL (1 volume) of ethanol (≥ 95%) to your sample and mix by pipetting or flicking the tube. Do not vortex. This will enable the binding of RNA ≥ 25 nt. If you wish to bind RNA as small as 15 nt, add 2 volumes (300 μL) of ethanol to your sample instead of 1 volume (150 μL). The addition of 2 volumes of ethanol shifts the cutoff size of RNA binding from 25 nt down to 15 nt.
  3. Insert column into collection tube, load sample onto column and close the cap. Spin for Duration00:01:00 , then discard flow-through. For diluted samples > 900 μL, load a portion of the sample, spin, and then repeat as necessary.
  4. Re-insert column into collection tube. Add Amount500 µL Cleanup Wash Buffer and spin for Duration00:01:00 . Discard the flow-through.
  5. Repeat wash (Step 4).
  6. Transfer column to an RNase-free 1.5 mL microfuge tube (not provided). Use care to ensure that the tip of the column does not come into contact with the flow-through. If in doubt, re-spin for Duration00:01:00 to ensure traces of salt and ethanol are not carried over to the next step.
  7. Elute in Amount20 µL nuclease-free water. The eluted RNA can be used immediately or stored at Temperature-80 °C . Care should be used to ensure the elution buffer is delivered onto the matrix and not the wall of the column to maximize elution efficiency.
3m
Lopez Lab Specifics: Characterization: To check QC (size and degradation) and endotoxins.
Lopez Lab Specifics: Characterization: To check QC (size and degradation) and endotoxins.
For QC
https://research.wustl.edu/core-facilities/genome-technology-access-center/
Person in charge: Michael Heinz mheinz@wustl.edu
4444 Forest Park Ave. 04140
Online submission form: https://gtac-old.wustl.edu/request/service/index.php
Quantity: 30 ng in 10 μL nuclease free H2O.
Shipment: 1.7-2.0 mL microcentrifuge tubes / dry ice

For Endotoxins
https://research.wustl.edu/core-facilities/biologic-therapy-core-facility-siteman-cancer-center/
Person in charge: Kelsi Rotter krotter@wustl.edu
Southwest Tower 00719
Quantity: 500 ng in 150 μL nuclease free H2O.
Shipment: 1.5 mL microcentrifuge (new plasticware certified to be pyrogen free) / dry ice