<aside> <img src="/icons/light-bulb_gray.svg" alt="/icons/light-bulb_gray.svg" width="40px" />

Getting Started

</aside>

Transfer RNAs (tRNAs) are small RNA molecules (76 nt - 90 nt) that carry amino acids to ribosomes during protein synthesis. They are essential for translation and can be easily purified following this protocol, which covers bacterial growth, purification, dialysis, and quality assurance. You can use these tRNAs in Make Energy Mix.

<aside> <img src="/icons/wrench_gray.svg" alt="/icons/wrench_gray.svg" width="40px" />

Materials and Equipment

</aside>

Reagents

‼️All reagents and materials must be prepared RNase-free. Use RNaseZap or 10% bleach to decontaminate plastic and glassware and rinse with nuclease-free water. We find ultrapure water (18.2 MOhm) is often sufficient for RNase-free work.

• Table 1. Reagents info: manufacturer, catalog #, pricing, & storage conditions

⚠️ Hazardous Materials

• Acid Phenol

• Acetic Acid

• Chloroform

• Ethanol

• Table 2. Materials info: manufacturer, catalog #, pricing & storage conditions

Buffers

• Table 3. Buffer concentrations, required volume, and storage conditions
• Table 4. Extraction Buffer Recipe

<aside> <img src="/icons/iterate_gray.svg" alt="/icons/iterate_gray.svg" width="40px" /> Protocol

</aside>

• [ ] Cell culture:
  • [ ] Prepare overnight cultures.
    • Notes - We purify tRNAs from A19
    • [ ] Add 6 mL Luria Broth (LB) under sterile conditions to two (2) 14 mL culture tubes. 6 mL is enough to innoculate 4 x 900 mL bulk outgrowths.
    • [ ] Label one tube “(+)”. Add 10 uL of MRE600 glycerol stock to (+).
    • Notes - you can work from glycerol stocks OR colonies.
    • [ ] Label the other tube “(-)”. This will be your negative control, testing if your technique is sterile.
    • [ ] Incubate both tubes overnight shaking at 37C / 225 - 250 rpm / 10 - 16 hr.
  • [ ] Perform bulk outgrowth.
    • [ ] Check if (-) has growth. If not, continue.
    • [ ] Back dilute overnight 1:500 - 1:1000 into 4x 900 mL fresh LB in 2 L baffled Erlenmeyer flasks (e.g., 900 uL overnight into 900 mL LB).
    • [ ] Incubate back diluted cultures at 37C / 225-250 rpm to mid-log phase (OD600 between 0.6 and 0.8). This took us ~3 hrs.
  • [ ] Pellet, wash, and store cells.
    • [ ] Fill 500 mL centrifuge bottles with culture. Balance centrifuge bottles against each other and centrifuge cultures at 16 000 rcf / 4C / 10 min.
    • [ ] Decant supernatant, add fresh culture, and repeat centrifugation as above, working through the remaining culture. You should end up with large pellets at the bottom of each centrifuge bottle.
    • [ ] Wash the pellets by resuspending in 500 mL cold (4C) NaCl (0.9%) then re-pelleting at 16 000 rcf / 4C / 10 min.
    • [ ] Transfer pellets by spatula into a tared bag weigh and record the mass.
    • [ ] Flash freeze pellet in liquid nitrogen and store at -80C.
• [ ] First Nucleic Acid Extraction:
  • [ ] Set centrifuge to 4C and set shaking incubator to 37C.
  • [ ] Resuspend 2 g of biomass into 18 mL of Extraction Buffer: NaOAc (50 mM), Mg(OAc)2 (10 mM), pH 5.0 in a 50 mL centrifuge tube by vortexing.
  • [ ] Add 18 mL of Acid Phenol (pH 4.5) using a glass serological pipette in a fume hood.
  • [ ] Cap the 50 mL centrifuge tube and seal with parafilm to prevent spillage.
  • [ ] Incubate at 37C / 225 rpm / 30 min in a shaking incubator. Tape tubes against the bottom plate of the shaking incubator horizontally so that samples are shaking laterally.
  • [ ] Centrifuge at 4000 rcf / 4C / 15 min. You should observe three (3) layers: the aqueous (top) fraction, the organic (lower) fraction, and a middle fraction of cell debris separating them.
  • [ ] Carefully collect the aqueous fraction by serological pipette, without disturbing the cell debris fraction, and transfer to a fresh 50 mL centrifuge tube.
• [ ] Second Nucleic Acid Extraction:
  • [ ] Add 14 mL of Extraction Buffer to Acid Phenol, parafilm the 50 mL centrifuge tube, and incubate at 37C / 225 rpm / 15 min.
  • [ ] Centrifuge at 4000 rcf / 4C / 15 min.
  • [ ] Collect the aqueous fraction and combine with the first nucleic acid extraction (total volume between 30 mL and 32 mL).
• [ ] Phenol Clean-up:
  • [ ] Split aqueous layer into 2x 50 mL centrifuge tubes and add one volume chloroform (~16 mL) to each tube using a glass serological pipette. Vortex for 5 minutes at RT.
  • [ ] Centrifuge at 4000 rcf / 4C / 15 min. You should see two (2) fractions: the aqueous (top) fraction and the organic (lower) fraction. The aqueous fraction should be more clear after washing with chloroform.
  • [ ] Collect the aqueous fraction and transfer to a fresh 50 mL centrifuge tube.
• [ ] Precipitate Nucleic Acids (RNA & DNA):
  • [ ] Set centrifuge to 25C.
  • [ ] Add NaCl (5 M) to the aqueous phase to a final concentration of 0.2 M (~1.5 mL). Mix by inversion and split evenly into 2x 50 mL centrifuge tubes.
  • [ ] Precipitate nucleic acids by adding one volume of isopropanol (~17 mL) to each tube and incubate at RT for 10 min. The mixture should turn cloudy.
  • [ ] Pellet nucleic acid precipitate via centrifugation at 14 500 rcf / 25C / 15 min.
  • [ ] Wash the pellet with EtOH (70%):
    • [ ] Decant supernatant and wash nucleic acid pellet with 10 mL cold (-20C) EtOH (70%). Break apart pellet by pipetting or vortexting to ensure salt is removed.
    • [ ] Re-pellet nucleic acid pellet by centrifugation at 14 500 rcf / 25C / 5 min.
    • [ ] Decant the supernatant and allow the pellet to air dry for 10 minutes.