Overview¶
Transfer RNAs (tRNAs) are small RNA molecules (76 - 90) nt that carry amino acids to ribosomes during protein synthesis. They are essential for translation and can be readily purified following this protocol, which covers bacterial growth, purification, dialysis, and quality assurance.
Important Information
Please read this section carefully. It contains important notes, resources, and safety information. Not all information included here is included in the lab-ready protocol.
Notes
We purify tRNAs and ribosomes from E. coli A19 biomass (CGSC 5997), a strain with a mutation in RNase I that improves RNA yields. You can use other RNase deficient strains with this protocol, but you may need to optimize this protocol to achieve high yield and purity.
‼️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 sufficiently RNase free without treatment.
Hazardous Materials
Acid Phenol
Corrosive, toxic, rapidly absorbed through skin, & respiratory irritant
Use in fume hood, wear neoprene gloves, lab coat, and goggles.
Acetic Acid
Corrosive to skin and eyes
Use appropriate PPE and handle under fume-hood
Ethanol
Highly flammable, toxic, and irritant
Wear PPE, use in well-ventilated areas, and keep away from open flames
Composition
| Component | Stock Concentration | Reaction Concentration |
|---|---|---|
| tRNA | 35 g/L (10x) | 3.5 g/L |
Materials and Equipment¶
Bill of Materials
| Name | Category | Product | Manufacturer | Part # | Price | Storage | Link |
|---|---|---|---|---|---|---|---|
| Sodium Acetate | Salts | Sodium acetate trihydrate,ACS reagent, ≥99% | Sigma-Aldrich | 236500-500G | $87.90 | 4°C to 30°C | link |
| Magnesium Acetate | Salts | Magnesium acetate tetrahydrate | Sigma-Aldrich | M0631-100G | $34.80 | 4°C to 30°C | link |
| Sodium Chloride | Salts | Sodium Chloride, Redi-Dri™, anhydrous, free-flowing, ACS, ≥99% | Sigma-Aldrich | 746398-1KG | $133.00 | 4°C to 30°C | link |
| Acetic Acid (glacial) | Salts | Acetic Acid, Glacial (Certified ACS), Fisher Chemical | Fisher Scientific | A38-212 | $458 | 4°C to 30°C | link |
| Acid Phenol (pH 4.5) | Supplements | Invitrogen ambion Acid Phenol:Chloroform, pH 4.5 (with IAA, 125:24:1) | Fisher Scientific | AM9722 | $313.65 | 2°C to 8°C | link |
| Isopropanol | Supplements | BioReagent 2-Propanol, Molecular Biology Grade, Liquid, ≥99.5% | Sigma-Aldrich | I9516-1L | $142.00 | 4°C to 30°C | link |
| Ethanol | Supplements | Ethanol, HPLC/Spectrophotometric Grade, Liquid, ≥99.5%, Glass bottle, 200 Proof | Sigma-Aldrich | 459828-4L | $493.00 | 4°C to 30°C | link |
| SYBR-Green stain | Supplements | Invitrogen sybr Green I Nucleic Acid Gel Stain 10,000x concentrate in DMSO | Fisher Scientific | S7567 | $791.70 | -25°C to -15°C | link |
| 2x TBE-Urea sample buffer | Supplements | Invitrogen novex TBE-Urea Sample Buffer (2x) | Thermo Scientific | LC6876 | $75.60 | 2°C to 8°C | link |
| NEB low range ssRNA ladder | Supplements | Low Range ssRNA Ladder - 100 gel lanes | New England Biolabs | N0364S | $87.00 | -25°C to -15°C | link |
| LB | Media | Luria Broth (Miller’s LB Broth), Non-Sterile, pH 6.8-7.2, Molecular Biology Grade, Powder | Sigma-Aldrich | L3522-1KG | $221 | 4°C to 30°C | link |
| A19 E. coli | Strains | A19 (RNase I mutant) | CGSC | 5997 | $130.00 | -80°C | link |
| Culture tubes (14 mL) | Consumables | 14mL Culture Tube and Dual Cap, PP, Sterile | CELLTREAT | 230439 | $190.00 | 4°C to 30°C | link |
| 2 L baffled Erlenmeyer flasks | Consumables | PYREX® 2L Delong Shaker Erlenmeyer Flask with Baffles | Corning | 4444-2L | $96.44 | 4°C to 30°C | link |
| 1 L centrifuge bottles | Consumables | Thermo Scientific™ # Fiberlite 1000mL Bottles | Thermo Scientific | 010-1491 | $326.84 | 4°C to 30°C | link |
| 50 mL centrifuge tubes | Consumables | Corning® 50 mL Polypropylene Centrifuge Tubes, Sterile, Racked, CentriStar™ Cap | Corning | 430828 | $436.88 | 4°C to 30°C | link |
| 15 mL centrifuge tubes | Consumables | Corning® 15 mL PP Centrifuge Tubes, Rack Packed with CentriStar™ Cap, Sterile, 50/Rack, 500/Case | Corning | 430790 | $230.44 | 4°C to 30°C | link |
| Slide-A-Lyzer G3 Dialysis Cassettes (3.5 kDa) | Consumables | Slide-A-Lyzer™ G3 Dialysis Cassettes, 3.5K MWCO | Thermo Scientific | A52966 | $128.70 | 4°C to 30°C | link |
| Amicon Ultra 0.5 mL - 3 kDa | Consumables | Amicon® Ultra Centrifugal Filter, 3 kDa MWCO | Millipore Sigma | UFC5003 | $63.00 | 4°C to 30°C | link |
| TBE-Urea 10% gel | Consumables | 10% Mini-PROTEAN | Bio-Rad | 4566033 | $41.00 | 2°C to 8°C | link |
| glass serological pipettes | Consumables | PYREX® 10 mL Glass Serological Pipets, Sterile, Individually Paper/Plastic Wrapped, Plugged | Corning | 7077-10N | $690.49 | 4°C to 30°C | link |
Protocol¶
Prepare Stock Buffers¶
Make the following stock solutions from solid. Use ultrapure water (18.2 MΩ, e.g., Milli-Q) and keep everything RNase-free.
| Stocks | Final Concentration (mM) | MW (g/mol) | Mass to add (g) | Final Vol (mL) | Storage (°C) |
|---|---|---|---|---|---|
| Sodium Acetate | 1000 | 136.08 | 136.08 | 1000 | room temp |
| Magnesium Acetate | 1000 | 214.46 | 214.46 | 1000 | room temp |
| NaCl (5 M) | 5000 | 58.44 | 292.2 | 1000 | room temp |
| NaCl (1 M) | 1000 | 58.44 | 11.688 | 200 | room temp |
Prepare Buffers¶
Adjust pH to 5.0 with glacial acetic acid, then bring to final volume with water.
Extraction Buffer — used to resuspend biomass for acid-phenol extraction.
| Reagent | Final Concentration (mM) | Stock Concentration (mM) | Volume to Add (mL) |
|---|---|---|---|
| Sodium Acetate | 50 | 1000 | 50 |
| Magnesium Acetate | 10 | 1000 | 10 |
| Acetic Acid (glacial) | — | — | to pH 5.0 |
| Ultrapure water | — | — | to 1000 |
| Total | 1000 |
NaOAc (300 mM, pH 5.0) — used to dissolve the nucleic-acid pellet before DNA removal.
| Reagent | Final Concentration (mM) | Stock Concentration (mM) | Volume to Add (mL) |
|---|---|---|---|
| Sodium Acetate | 300 | 1000 | 30 |
| Acetic Acid (glacial) | — | — | to pH 5.0 |
| Ultrapure water | — | — | to 100 |
| Total | 100 |
Prepare Working Buffers¶
Make this wash solution as needed; dilute from ~100% (200 proof) ethanol.
Ethanol (70% v/v) — used to wash nucleic-acid pellets during precipitation.
| Reagent | Final Concentration | Stock Concentration | Volume to Add (mL) |
|---|---|---|---|
| Ethanol | 70% (v/v) | 100% (v/v) | 700 |
| Ultrapure water | — | — | 300 |
| Total | 1000 |
Cell culture¶
Note: you can work from glycerol stocks OR colonies.
We first need to prepare bacterial cultures. We will work from 6 mL overnight cultures of our expression strains and backdilute them the next day. In order to prepare these overnight cultures, we need stocks of bacteria.
We work with 100 µL aliquots of our glycerol stocks frozen in PCR strip tubes. When seeding our overnights with bacteria, we poked one glycerol aliquot with a pipette tip and ejected the tip into culture tubes (more details below).
Optionally, you can work from individual colonies by streaking out your bacterial stocks. Working from colonies assures that your bulk outgrowth will have come from a single colony forming unit.
Prepare overnight cultures.
Add 3 mL Luria Broth (LB) under sterile conditions to three (3) 14 mL culture tubes. Two (2) tubes will be used to prepare 6 mL of overnight culture and one (1) tube will be used as a negative control. 6 mL of overnight culture is enough to inoculate 4 x 450 mL of bulk outgrowths.
Label two tubes “(+)” and seed with an A19 stock (colony or glycerol stock; see note above).
Label the other tube “(-)”. This will be your negative control, used to test your sterile technique.
Incubate all tubes overnight at 37°C / (225 - 250) rpm / (10 - 16) hr.
Perform bulk outgrowth.
Check if (-) has growth. If not, continue.
Seed 4x 450 mL fresh media in 4x 2L baffled Erlenmeyer flasks with 1:500 overnight culture (e.g., 900 µL overnight into each flask with 450 mL media).
Incubate back diluted cultures at 37°C / (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 1 L centrifuge bottles with culture. Balance centrifuge bottles against each other and pellet cultures at 16 000 rcf / 4°C / 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 (4°C) NaCl (0.9%) then pelleting again at 16 000 rcf / 4°C / 10 min.
Transfer pellets by spatula into a tared bag weigh and record the mass.
Flash freeze pellet in liquid nitrogen and store at -80°C.
Nucleus acid extraction by precipitation¶
First Nucleic Acid Extraction:
Set centrifuge to 4°C and set shaking incubator to 37°C.
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.
In a fume hood and wearing the appropriate PPE, add 18 mL of Acid Phenol (pH 4.5) using a glass serological pipette.
Cap the 50 mL centrifuge tube and seal with parafilm to prevent your sample spilling.
Incubate at 37°C / 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 / 4°C / 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, seal the 50 mL centrifuge tube with parafilm, and incubate at 37°C / 225 rpm / 15 min.
Centrifuge at 4000 rcf / 4°C / 15 min.
Collect the aqueous fraction and combine with the first nucleic acid extraction (total volume between 30 mL and 32 mL).
Precipitate Nucleic Acids (RNA & DNA):
Set centrifuge to 25°C.
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 room temperature for 10 min. The mixture should turn visibly cloudy.
Pellet nucleic acid precipitate via centrifugation at 14 500 rcf / 25°C / 15 min.
Wash the pellet with EtOH (70%):
Decant supernatant and wash nucleic acid pellet with 10 mL cold (-20°C) EtOH (70%).
Re-pellet nucleic acid pellet by centrifugation at 14 500 rcf / 25°C / 5 min.
Decant the supernatant and allow the pellet to air dry for 10 min.
Remove rRNA by precipitation:
Resuspend each pellet into 15 mL of cold (4°C) NaCl (1 M) by vortexing or pipetting. Ensure the pellet is fully dissolved. Allow NaCl (1 M) solution to hydrate pellet for (10 - 15) min at room temperature to help the pellet dissolve.
Precipitate rRNA by centrifugation at 9500 rcf / 4°C / 20 min.
Decant the supernatant to a new 50 mL centrifuge tube.
Precipitate remaining DNA and tRNA nucleic acids:
Add 2 volumes (approximately 30 mL) of cold (-20°C) EtOH (100%) to the supernatant and incubate at -20°C / >30 min to precipitate remaining nucleic acids. You can perform this step overnight.
Centrifuge at 14 500 rcf / 4°C / 5 min.
Wash the pellet with EtOH (70%) as above.
Remove DNA by precipitation:
Set centrifuge to 25°C.
Dissolve the pellet in 6 mL of NaOAc (300 mM, pH 5.0). As needed to ensure the pellet is fully dissolved, heat samples up to 60°C, pipette mix, and/or vortex. If the pellet is visibly small, you can dissolve each pellet in 3 mL of NaOAc (0.3 M, pH 5.0) and pool them together, totaling 6 mL.
Add 0.56 volumes of isopropanol (~3.4 mL) to each nucleic acid solution and incubate at room temperature for 10 min.
Centrifuge at 14 500 rcf / 25°C / 5 min. Decant the supernatant to a 15 mL centrifuge tube.
Precipitate tRNAs:
Set the centrifuge to 4°C.
Add 2.3 mL of isopropanol to the supernatant (supernatant:isopropanol is 100:95) and incubate at -20°C / >30 min. This step can be performed overnight.
Centrifuge the suspension at 14 500 rcf / 4°C / 15 min.
Wash the pellet with EtOH (70%) as above.
Resuspend the tRNA pellet in 1.5 mL of nuclease-free water and keep on ice.
Dialysis¶
Note: we use dialysis cassettes
We typically use dialysis cassettes rather than dialysis membranes for ease of use. Instructions below are for dialysis cassettes, but should work for either. Use membranes with a molecular weight cut off < 10 kDa (we use 3.5 kDa). See # Slide-A-Lyzer™ G3 Dialysis Cassettes, 3.5K MWCO for an example commercial dialysis cassette.
Hydrate the dialysis membrane: Remove the cassette from its protective pouch and immerse in nuclease-free water for 2 min.
Add Sample:
Open the cassette by twisting the cap counter-clockwise.
Carefully pipette 1.5 mL of resuspended tRNAs into the cassette. Avoid puncturing the membrane!
Remove the excess air in the cassette by simultaneously pressing the membrane gently on both sides and inserting the cap and locking it into place.
Dialyze Sample:
Float cassette in 500 mL nuclease-free water in a large (>600 mL) beaker and gently stir at 4°C / 2 hrs. We do this by putting our beaker in a bucket of ice on a stir plate.
Change the dialysis buffer and continue dialyzing overnight.
Concentrate¶
Pipette dialyzed tRNAs to the upper chamber of an Amicon® Ultra-0.5 mL Centrifugal Filter, 3 kDa MWCO.
Centrifuge at 14 000 rcf / 10 min and check the remaining volume in the upper chamber. Repeat until you hit your target volume.
Note: estimate your target volume at 100 µL per 1 g biomass.
From our experience, a good rule of thumb is to target a final volume around 100 µL per 1 g of biomass used (e.g., 3.6 gDCM → concentrate to ~360 µL (~40 µg/µL tRNAs)).
Quality control¶
Estimate concentration by UV-Vis Spectroscopy (Nanodrop).
Prepare a 1:1000 dilution of your tRNAs in water.
Measure absorbance at 260 nm, 280 nm, and 230 nm.
Estimate your yield by A₂₆₀ ([tRNA] = A₂₆₀ * 40 mg/mL). Typical yield of tRNA is typically between (4-20) mg per gram of cell mass.
Estimate your purity by A₂₆₀/A₂₃₀ and A₂₆₀/A₂₈₀ (both should be ≥1.8).
Note: [tRNA] = A₂₆₀ * 40 mg/mL
A₂₆₀ units (a.k.a. “absorbance units”, or “A₂₆₀”) are defined as the amount of light (wavelength = 260 nm) absorbed as it passes through 1 cm of the sample being measured.
This value is measured by diluting a sample by some factor such that the measured absorbance is in the linear range of your device (A₂₆₀=0.5 is typically acceptable), then multiplying the measured absorbance by that dilution factor.
These units are difficult to think about, but easy to measure. If you want to report mass concentration of your samples instead, use the following approximate conversion: 40 mg/mL ~ A₂₆₀ units.
NEB has a great tool for converting absorbance units to concentrations. Make sure to select “ssRNA”.
Visualize purity by TBE-Urea 10% Gel.
(optional) Prepare TBE-Urea 10% Gel
Load gel into gel dock with running buffer.
Pre-run gels at 100 V / 30 min.
Wash wells with running buffer by syringe. You should be able to see urea displaced from the well by change in refractive index.
Prepare tRNA samples:
Dilute tRNAs to 40 ng/µL tRNA in nuclase-free water.
Prepare 20 ng/µL sample by adding 10 µL of 40 ng/µL tRNA to 10 µL 2x TBE-Urea sample buffer.
Prepare an ssRNA ladder. We use the NEB low range ssRNA (2 µL ladder + 2 µL 2x sample buffer).
Incubate sample and and ladder at 65°C / 3 min → 4°C / hold using a thermal cycler.
Load 200 ng of tRNA (10 µL at 20 ng/µL) onto the TBE-Urea gel and run at 125V / 2.5 hr.
Meanwhile, prepare SYBR-Green stain (4 µL in 40 mL water) to stain gel.
Soak gel in SYBR-Green stain and visualize gel using UV or blue-light transilluminator. You should see multiple distinct bands around 75-90 nt.
Note: RNases digesting your tRNA → smear on gel
If your sample is contaminated with RNases, your sample quality will decrease as the RNases degrade the tRNAs. A clear sign of tRNA degradation are poorly defined, smeared bands. A clean tRNA prep should have distinct bands.
If your samples appear as a smear on your gel, consider testing your input buffers and final prep for RNase activity. You can use the Invitrogen RNaseAlert™ Lab Test Kit (Cat. No. AM1964). We recommend diluting your tRNAs by adding 2 µL to 40 µL of provided sample buffer.
Formulation¶
Dilute your tRNA stocks in nuclease-free water to 35 µg/µL, which is 10x working concentration for cytosol reactions.
Storage¶
Aliquot your tRNAs to reduce freeze / thaw cycles and store at -80°C.