Editing In vitro capped mRNA

== In Vitro Transcription with MessageMax T7 ARCA-Capped Message Transcription Kit ==

1. Thaw RNase-free Water, 10X Transcription Buffer, ARCA Cap/NTP PreMix, 100 mM DTT, and plasmid DNA template, and centrifuge the tubes briefly. Warm these reagents to room temperature before assembling the reactions.

2. Set up an in vitro transcription reaction for the plasmid construct. Add the reaction components into a clean 1.5-ml microcentrifuge tube at room temperature in the order given below:<br>RNase-free Water 10 μl<br>Plasmid DNA template (~1μg/μl) 2 μl<br>10X Transcription Buffer 4 μl<br>ARCA Cap/NTP PreMix 16 μl<br>100 mM DTT 4 μl<br>MessageMax T7 Enzyme Solution 4 μl<br>Total reaction volume 40 μl<br>Incubate at 37 °C for 30 minutes.

Note: Extending the incubation time to 1 hour may increase the RNA yield in some instances.

3. At the end of the in vitro transcription incubation, centrifuge the tube briefly and add 2 μl of DNase I. Mix gently and<br>centrifuge briefly. Note: Do not vortex the DNase I or the DNase I digestion.

4. Incubate the DNase I digestion at 37 °C for 15 minutes.<br> Note: At the end of the DNase digestion, you may immediately proceed to the poly(A) tailing reaction or store the reaction at -20 °C overnight before proceeding to poly(A) tailing.





== Poly(A) Tailing with Poly(A) Polymerase Tailing Kit ==

1. Add the reaction components into a clean 1.5-ml microcentrifuge tube at room temperature according to the order given below:<br>RNase-free Water 109 μl<br>10X Reaction Buffer 20 μl<br>10 mM ATP 20 μl<br>ScriptGuard RNase Inhibitor (40 U/μl) 5 μl<br>In vitro transcription reaction 42 μl<br>Poly(A) Polymerase 4 μl <br>Total volume 200 μl 

2. Incubate the reaction at 37 °C for 1 hour.<br>Note: At the end of the poly(A) tailing reaction, you may proceed immediately to RNA purification or store the reaction at -20 °C overnight before purification.



== RNA Purification with MegaClear Kit ==

1. Add 700 μl of Binding Solution Concentrate to the poly(A) tailing reaction (200 μl) and mix thoroughly by vortex.

2. Add 500 μl of 100% ethanol and mix thoroughly by vortex.

3. Insert a Filter Cartridge into a Collection Tube and add 700 μl of the RNA sample into the Filter Cartridge

4. Centrifuge at ~16,000 x g for 30 seconds. Decant the flow-through liquid and return the Filter Cartridge to the Collection Tube.<br> Note: Centrifugation in this and all subsequent steps is performed at room temperature.

5. Add the remaining RNA sample into the same Filter Cartridge and centrifuge at ~16,000 x g for 30 seconds. Decant the flow-through liquid and return the Filter Cartridge to the Collection Tube.

6. Add 500 μl of alcohol diluted Wash Solution to each Filter Cartridge and centrifuge at ~16,000 x g for 30 seconds. Decant the flow-through <br>liquid and return the Filter Cartridge to the Collection Tube.

7. Add another 500 μl of alcohol diluted Wash Solution to each Filter Cartridge and centrifuge at ~16,000 x g for 30<br>seconds. Decant the flow-through liquid and return the Filter Cartridge to the Collection Tube.

8. Dry the Filter Cartridge by centrifugation at ~16,000 x g for 1 minute.

9. Transfer the Filter Cartridge into a new Collection Tube. 

Add 50 μl of Elution Solution directly onto the center of the filter inside the Filter Cartridge. Close the cap of the tube<br>immediately.

10. Heat the tube at 65 °C for 10 minutes on a clean heat block or in a clean water bath.<br>11. Centrifuge the tube at ~16,000 x g for 1 minute to elute RNA.

== Determination of RNA quantity and quality<br> ==

Measure the RNA concentration and the A 260/A 280 ratio of the diluted sample with a spectrophotometer or a NanoDrop instrument. When a spectrophotometer is used, RNA concentration and yield can be calculated as follows:

RNA concentration: A 260 x dilution factor x 40 = μg/ml<br>RNA yield: 0.05 ml x RNA concentration (μg/ml) = μg<br>RNA yield should be ≥ 40 μg per transcription reaction.<br>A 260/A 280 should be ≥ 2.2.<br><br>

[[Category:Protocols]]