Difference between revisions of "Team:Toronto/Experiment-S30 Protocol"

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Revision as of 06:20, 19 October 2016

S30 Protocol · Benchling

S30 Protocol

Introduction

Get started by giving your protoücol a name and editing this introduction.

Materials

  • 2 × YTPG
    • 1L Milli-Q Water
    • 2.99 g KH2 PO4
    • 6.97 g K2 HPO4
    • 19.82 g glucose
    • 16 g tryptone
    • 10 g yeast extract
    • 5 g NaCl
  • Buffer A:
    • 10 mM Tris-acetate (pH 8.2)
    • 14 mM magnesium acetate
    • 60 mM potassium glutamate, and
    • 2 mM dithiothreitol (DTT) or 2mM TCEP
  • Buffer B:
    • 1.2 mM ATP;
    • 0.85 mM each of GTP, UTP, and CTP;
    • 34.0 μg mL−1 L-5-formyl-5, 6, 7, 8-tetrahydrofolic acid (folinic acid);
    • 170.0 μg mL−1 of E. coli tRNA mixture;
    • 130 mM potassium glutamate;
    • 10 mM ammonium glutamate;
    • 12 mM magnesium glutamate;
    • 2 mM each of 20 amino acids;
    • 0.33 mM nicotinamide adenine dinucleotide (NAD);
    • 0.27 mM coenzyme-A (CoA);
    • 1.5 mM spermidine;
    • 1 mM putrescine;
    • 4 mM sodium oxalate;
    • 33 mM phosphoenolpyruvate (PEP);
    • 100 μg mL−1 T7 RNA polymerase

Procedure

  • Preparing the Extract:
  1. Grow E. coli BL21 in 2 × YTPG media:
  • (shake baffled flasks in a 37°C incubator with vigorous shaking at 250 rpm)
  1. Monitor by spectrophotometry until OD600 of 3.0 is reached (in the middle of exponential growth)
  1. Harvest the cells by centrifuging at 5000 x g at 4°C for 15 min
  1. Wash cells three with cold Buffer A (20mL for each gram of wet cells)
  1. Optional: After final wash and centrifugation, weigh and flash freeze in liquid nitrogen, and store at −80°C.
  1. [Re]suspend the pellets in 1 mL of Buffer A per 1 g of wet cell mass
  1. Transfer into 1.5 mL microtube and placed in an ice-water bath to minimize heat damage during sonication
  1. Llyse the cells at frequency of 20 kHz and 50% of amplitude. The input energy should add up to 556 Joules.
  1. Centrifuge the lysate at 12,000 RCF at 4°C for 10 min.
  1. Optional: incubate the supernatant at 37°C for 60 min with gentle shaking (250 rpm) and centrifuged at 15,000 RCF at 4°C for 15 min.
  1. Flash freeze in liquid nitrogen and stored at −80°C until use.
  • Reaction Mixture
  1. Add the following components to nuclease-free water to make 55 mL of 2 X Buffer B:
  • 0.005 g Folinic acid
  • 0.026 g E. coli tRNA mixture
  • 3.612 g Potassium glutamate
  • 0.025 g Ammonium glutamate
  • 0.305 g Magnesium glutamate
  • 0.039 g Isoleucine
  • 0.039 g Leucine
  • 0.044 g Lysine
  • 0.045 g Methionine
  • 0.050 g Phenylalanine
  • 0.036 g Threonine
  • 0.061 g Tryptophan
  • 0.035 g Valine
  • 0.052 g Arginine
  • 0.047 g Histidine
  • 0.027 g Alanine
  • 0.040 g Asparagine
  • 0.040 g Aspartate
  • 0.036 g Cysteine
  • 0.044 g Glutamate
  • 0.023 g Glycine
  • 0.035 g Proline
  • 0.032 g Serine
  • 0.054 g Tyrosine
  • 0.033 g NAD
  • 0.033 g Spermidine
  • 0.013 g Putrescine
  • 0.080 g Sodium oxalate
  • 0.832 g PEP
  • 0.015 g T7 RNA Polymerase
  1. Reactions are carried out in a 1.5 mL microtube in the incubator at 37°C for 4 hours
  1. The standard reaction mixture consists of the following components in a final volume of 15 μL:
  • 5.5 μL of 2X Buffer B
  • 200 ng plasmid
  • 4 μL of cell extract.
  • 1.275 μL of 10mM NTP mix
  • 1.485 μL of 2mM CoA (serial dilute 4μL of 50mM CoA in 96μL of ddH2O)
  • 0.35mM ATP (How much is this?)
  • top off with milliQ water to get to 15 μL



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