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Quick Video Tutorials for Next Generation Cell Free Protein Expression Kit (Wheat Germ) – CFPS700

How to Perform Transcription, mRNA Purification, and Translation

The following videos provide quick tutorials on the three major steps following PCR when using the Next Generation Cell Free Protein Expression Kit (Wheat Germ) (CFPS700): transcription, mRNA purification, and translation. For more details, please see the complete protocol.

How to prepare the transcription reaction
 

  1. Add RNase free water.
  2. Add Transcription Buffer.
  3. Add NTP mix.
  4. Add DTT.
  5. Add T7 RNA Polymerase.
  6. Finally, add 2nd PCR product.
  7. Mix the solution with tapping lightly.
  8. Drop the solution on the tube wall with a tabletop centrifuge.
  9. Transcription reaction is completed by putting in water bath or incubator at 37°C for 3 hours.


How to purify the mRNA product
 

  1. Add 4M ammonium acetat to the solution after completion of the transcription reaction.
  2. Add 100% ethanol.
  3. Mix the solution with tapping lightly.
  4. Drop the solution on the tube wall with a tabletop centrifuge.
  5. Let it stand on ice for 20 minutes.
  6. Centrifuge at 15,000 rpm for 20 minutes.


How to prepare the translation reaction
 

  1. Centrifuge the mRNA solution then discard the supernatant.
  2. Centrifuge with tabletop centrifuge for several seconds. Discard the supernatant again very carefully.
  3. Please be careful to not touch the pellet of the sample.
  4. Leave the pellet until dries so dust and brushes do not get in.
  5. During pellet drying, prepare a translation solution.
  6. Prepare Wheat Germ Extract in a new tube.
  7. Add Amino Acid Mix.
  8. Leave the translation solution at room temperature.
  9. Add RNase free-water to the dried mRNA to make an mRNA solution.
  10. Leave for 10 minutes. Tap the tube and suspend by tabletop centrifuge.
  11. Suspend by tapping and tabletop centrifuge.
  12. Add translation solution at room temperature to mRNA solution.
  13. Re-suspend gently to prevent foaming.
  14. Translation reaction is completed by putting in incubator at 16°C overnight (10 hours or more).

Materials