Total tRNA are bulk purified small RNAs from E. coli. Total tRNA can be used directly in translation reactions.

Part	Size	Concentration
Nucleus Total tRNA	1 mg	33.5 µg/µL

What’s in the box?

Part	Storage	Amount	Concentration	Buffer
Nucleus Total tRNA	-80C	1 x 30 uL	33.5 µg/µL	Nuclease-free ultrapure water
plamGFP control plasmid	4C	1 x 30 uL	148 ng/µL	Nuclease-free ultrapure water
plamGFP protein standard	-80C	1 strip (8 x 25 uL)	0, 20, 50, 75, 100, 125, 150, 300 ng/uL	Nuclease-free ultrapure water

Quick tips:

Store at -80C!
Thaw tRNAs on ice immediately before use.
Mix by vortexing or pipette. ****
Avoid multiple freeze-thaw cycles (≤ 3).
Limit exposure to RNAses.

Product information

What is tRNA?

Transfer RNAs (tRNAs) are small RNA molecules (76-90 nt) that carry amino acids to ribosomes during protein synthesis. They are an essential component of in vitro translation reactions. Nucleus Total tRNAs are a mixture of co-purified tRNAs from E. coli MRE600.

Preparation

Nucleus Total tRNA is prepared from E. coli MRE600 biomass by warm Phenol Extraction. Briefly:

we prepare E. coli biomass from LB in shake flasks and grow out cultures to mid-log phase (OD600 between 0.6 and 0.8) and harvest by centrifugation.
Mixed nucleic acids (DNA and RNA) are extracted from biomass by gentle lysis with Acid Phenol (pH < 5), washed with chloroform, and precipitated with isopropanol in high salt (≥ 200 mM NaCl).
Large nucleic acids (e.g., gDNA and rRNA) are selectively precipitated in aqueous high salt (≥ 1M NaCl).
DNA is selectively precipitating in isopropanol in high salt (≥ 200 mM NaCl) at pH ≤ 5.
tRNAs are precipitated in cold isopropanol, washed, resuspended, and dialyzed against Ultrapure water.
tRNAs are concentrated to working volumes by centrifugal filtration and stored in Ultrapure water at -80C.

Nucleus Total tRNA is built using the Make tRNAs protocol.

Usage

Nucleus Total tRNA is shipped at 33.5 ug/uL in Ultrapure water. These tRNAs can be added directly to in vitro translation reactions without further preparation. For PURE reactions, use 1 uL Nucleus Total tRNA per 10 uL PURE reaction.

Thaw tRNAs on ice immediately before use. Mix by vortexing or pipette.

Store at -80C! Avoid multiple freeze-thaw cycles (≤ 3).

NOTE: limit exposure of Nucleus Total tRNA to RNAses.

Component	Volume per Rxn (uL)
NEB SolA dtRNA	2
NEB AA Mix	1
NEB-Sol B	3
NEB RNase Inhibitor	0.5
Reporter DNA (e.g.,`pT7-plamGFP`) @100 fmol/uL	0.5
Nucleus Total tRNA
Nuclease Free Water	1
Total	10