Apr 10, 2026
In-vitro Transcription of T7 Cas9 mRNA for Drosophila Embryo Injections
Forked from a private protocol
- 1Janelia Research Campus - HHMI;
- 2HHMI & Stowers Institute
Protocol Citation: Elizabeth Kim, David Stern 2026. In-vitro Transcription of T7 Cas9 mRNA for Drosophila Embryo Injections. protocols.io https://dx.doi.org/10.17504/protocols.io.kxygxjx2kl8j/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: April 10, 2026
Last Modified: April 10, 2026
Protocol Integer ID: 314832
Keywords: CRISPR , Cas9 mRNA, Drosophila, Transfection, Transformation, Transgenics, T7, Embyro Injections, In vitro, Transcription, mRNA, preparing t7 cas9 mrna, transcription of t7 cas9 mrna, t7 cas9 mrna, drosophila embryo injections this protocol, clean mrna sample for embryo injection, drosophila embryo injection, plasmids with t7 expression system, other transcription of other plasmid, t7 promoter, capped mrna, clean mrna sample, transcription, t7 expression system such as t7 phic31, other transcription, embryo injection, t7 expression system, drosophila, t7 phic31, including drosophila, cas9, other plasmid, plasmid
Abstract
This protocol is designed for generating and preparing T7 Cas9 mRNA for co-injection into Drosophila. In vitro synthesis of capped mRNA is utilized in co-injections of many systems including Drosophila. This protocol utilizes plasmids with T7 expression system with a T7 promoter. It provides a double cleaning method that ensures a clean mRNA sample for embryo injections. While it specifically refers to Cas9 it may be utilized for other transcription of other plasmids uitlizing the T7 expression system such as T7 phiC31 for attP and attB integration.
Guidelines
Please note this protocol, while it may be completed in one day, has been optimized for injection. The several day process allows for maximum concentrations at each step.
Materials
EcoRI-HF (New England BioLabs)
QIAquick PCR Purification Kit (Qiagen)
Qiagen Midi Prep Kit (Qiagen)
mMESSAGE mMACHINE™ T7 Transcription Kit (Invitrogen)
MEGAclear™ Transcription Clean-Up Kit (Invitrogen)
Before start
Before starting, clean surfaces and wipe down pipettes with RNA degradation solution such as RNAse Away.
Section 1: Midi Prep of plasmid
Step 1: Midi Prep of plasmid - Qiagen Midi Prep Kit
P508 T7-Cas9-nos-3'UTR
Prepare a culture of 100 mL to grow overnight.
Please refer to Qiagen’s midi prep kit for full protocol instructions.
Elute in 80 –ul of Elution Buffer or water. ( While the the protocol calls for 200 ul of Elution Buffer, using 80 -100 ul of Elution buffer will produce higher concentrated stock solution for linearization)
While preparing plasmid does not require midi prep, it is useful in getting large
amounts with higher concentration. The T7 protocol requires a high concentration
of linearized product, around 900 ng to 1.2 ug. Higher concentrations of
linearized product results in higher concentrations of mRNA.
Section 2: Linearization of the construct with EcoR1 digestion.
Mix according to table and split into 4 PCR tubes for 50 ul reaction for digestion
| A | B | |
| H2O | depends on DNA volume | |
| CutSmart Buffer | 20 ul | |
| Enzyme EcoRI-HF | 16 ul | |
| DNA | ~ 16 ug | |
| TOTAL | 200 ul Master Mix |
*Please note that the cut site EcoRI-HF is specific to the plasmid utilized here. Cut sites may vary depending on working construct.
Incubate overnight at 37oC
Inactivate at 65oC for 5 minutes.
Section 3: Purification of linearized DNA with Qiagen QIAquick PCR purification kit
Qiagen QIAquick PCR purification kit
Catalog # 28104
Combine two of the Linearized reaction in order to follow the Qiagen protocol in 2 columns
100 uL rxn + 500 uL PB
Elute in 12 - 15 uL total EB
Quantify with Qubit™ dsDNA Quantification Assay Kits Catalog number: Q32850
If too concentrated add more water to column and spin again to collect and increase
volume. The goal of this step is to obtain a concentration of around 200 ng/ul or higher
Gel Electrophoresis Check on with 1% gel to make sure complete digestion.
Far left figure : 1 KB Ladder utilized
Lane 1: 1 kb ladder
Lane 2: uncut plasmid (1ul of midiprep)
Lane 3: linearized plasmid pooled reaction 1 (1 ul)
Lane 4: linearized plasmid pooled reaction 2 (1 ul)
Note that any linearized product not used in the transcription process may be stored at 4oC for several months. If using a previously linearized DNA, make sure to run a second gel to confirm DNA remains linearized.
Section 4: In vitro transcription RXN: Capped mRNA production
Following the manual of mMESSAGE mMACHINE® T7 Ultra Kit (Page11-12) but in multiples of 4 reactions.
Catalog # AM1345 from Ambion/Life Technologies
Clean bench and wipe down area and pipettes with RNAse Away or RNAzap.
Thaw, vortex and spin down all reagents: 10X Reaction Buffer and 2X NTP/CAP.
Keep 2X NTP/ ARCA at 4oC and 10X Reaction Buffer at room temperature during assembly
Assemble the reactions into 1.5 ul tube
This is a master mix of 4 reactions (for different number of reactions see T7 protocol)
| A | B | |
| Amount | Component | |
| -varies | Water | |
| 40 ul | 2X NTP/ARCA | |
| 8 ul | 10X Reaction Buffer | |
| ~3,600 – 4,800 ng | Linear DNA | |
| 8 ul | T7 Enzyme Mix | |
| 80 ul | TOTAL VOLUME |
Mix and quick spin
Split the reaction into 4 PCR tubes and incubate at 37oC for 2 hours (DON’T CLOSE LID FOR INCUBATIONS : lid heating is necessary for iPCR but not for incubations)
Degrade DNA by adding 1.0 ul of Turbo DNase I to each tube . Incubate at 37oC for 15 minutes
During 15 minute incubation, Mix A tailing reagent:
This is a master mix of 4 reactions (for different number of reactions see T7 protocol)
| A | B | |
| Amount | Component | |
| 145 ul | Nuclease Free water | |
| 80 ul | 5X E-PAP Buffer | |
| 40 ul | 25 mM MnCl2 | |
| 40 ul | ATP Solution |
*added an extra ul of water to provide room for equal distribution of A tailing Master Mix (from 144 ul to 145 ul water)
Add 76 uL A-tailing reagents to each tube.
Bioanalyzer Step (optional)
Take a 1.5 uL aliquot from each PCR tube and pool into a separate PCR tube before adding A tailing to use as a tailing control. Pool 1-2 and 3-4
”before A-tailing”.
Add 4 μL E –PAP into each PCR tube mix gently (E-PAP enzyme facilitates A tailing)
Incubate at 37°C for 45 min. Place on ice.
Bioanalyzer Step (optional)
Take a 1.5 uL aliquot of each sample. Pool 1-2 and 3-4.
after A-tailing”
Stop the reaction and precipitate the RNA by adding 50 μL LiCl Precipitation Solution.
Pool 1-2 and 3-4 into two separate 1.5 ml eppendorf tube to avoid spilling. Mix thoroughly.
Chill O/N at –20°C.
Pre-chill a centrifuge to 2 - 4°C and Centrifuge at maximum speed for 30 to 45 minutes to pellet the RNA.
During centrifugation, make fresh 2 mL of 70% Ethanol using 200 proof ACS Ethanol
After centrifugation, leave one reaction in chilled centrifuge at 2-4°C while working
on other reaction for steps 17-21.
After centrifugation, check for presence of small white pellet. Should be a sizable amount.
Approximately this big o or larger. Anything smaller will probably yield small concentrations
Remove the supernatant without disturbing pellet.
Wash the pellet with the prepared ~1 mL 70% ethanol and re-centrifuge.
Remove the 70% ethanol
Resuspend the RNA in 100 uL of Elution Solution (from MEGAclear™ Transcription Clean-Up Kit)
When resuspend either vortex or pipette up and down until you can no longer see pellet.
Please note that the white pellet will turn translucent as it resuspends. It will be easier to see translucent pellet dissolve completely by pipetting up and down.
Section 5: Purify RNA with MEGAclear™ Transcription Clean-Up Kit
MEGAclear™ Transcription Clean-Up Kit
Catalog # AM1908 from Invitrogen
Add 350 ul Binding Solution to the 100 ul reaction and mix well
Add 250 ul 100% ethanol and mix well.
Apply the sample (700ul) in the Spin column and spin for 15s. Discard the flow-through.
Wash the column with 2 x 500 μL Wash Solution and spin for 15s. Discard the flow-through
Spin for an extra minute to get rid of any residual wash solution
Transfer columns/tubes to new tubes.
Apply 20 of nuclease free water. Close the tube and incubate at 65–70°C for 5–10 min, spin for 1
min.
*Make sure to elute with water and not elution buffer as this final product will be used as part of the injection solution for embryo injection.
Repeat with a second 20 μL of Elution Solution with another incubation period of 65 – 70 °C for
another 5 minutes.
Bioanalyzer Step (optional)
Take a 3 uL aliquot. “after A-tailing and cleaning (purify)” then take all three sample aliquots and the RNA ladder (~ 2 ul) and incubate at 90 °C for 2 minutes before loading on chip.
Qubit quantify using Qubit™ RNA Broad Range (BR) Assay Kit Catalog # Q10210
Typically concentrations for injections should be higher than 500 ng/ul so concentration amounts can be adjusted for injection solutions
Optional: Bioanalyzer Results Example
Lane 1: Ladder
Lane 2: Before A-tailing
Lane 3: After A-tailing
Sample 1: Before A-tailing
Sample 2: After A-tailing
Comments: The product looks good and final RNA concentration is 1.2 ug/μl, volume is 100 uL.