Basic Operations

Cultivation
  1. Write information such as gene, vector, strain name on a conical tube.
  2. Add 7 mLof liquid medium to the conical tube.
  3. Poke one colony on the place with a tip and drop the tip with colony into the liquid medhium.
  4. Shaking culturing at 37°C for 3 hours or 25°C for 12 hours.

Wastewater treatment

    Collect the medium containing recombinant E. coli in a Falcon or triangular flask covered with silicone plug, and place the medium in a bag as specified and then autoclave it to kill the bacteria. Dispose ethyl acetate and organic solvents discharged by the LCMS safety in accordance with the waste disposal regulations set forth by the university.

Autoclave
  1. Wrap aluminum foil around micropipette tips placed in a container, and place the medium in a container less than 1/3 of the container, and cover it with a silicone lid to ensure air permeability.
  2. Open the lid of the autoclave sterilizer (Tommy Seiko LBS-325) and pour tap water so that the liquid level is above the bottom.
  3. Place the items to be autoclaved in a mesh basket, and place the basket in the sterilizer.
  4. Close the lid tightly, start autoclaving at 121°C for 15 minutes, and wait for 90 minutes.
  5. Cool down the temperature of the can sufficiently, after that open the lid. And place the chips and other items to be dried more in the oven at 60°C for several hours.

Cloning

PCR, electrophoresis, and gel extraction
  1. Mix as follows. Modify the values according to the concentrations of the DNA or the template.
    compounds μL Final Concentration
    DW X
    KOD one Master Mix 12.5
    Primers 0.75 each 0.3 μM each
    Template Y Plasmid DNA: ~1ng/μL
    cDNA: 15ng/μL
  2. Amplify by adjusting the temperature in 2steps ((92°C/5sec→63°C/20sec)・30 ).
  3. Mix 1% agarose with TAE buffer, heat to dissolve, and solidify to form a gel, pour the sample into wells and electrophoresed at 100 V for 25 min.
  4. Cut out a thin slice of the desired band and extract DNA using the Japan Genetics FastGene™ Gel / PCR Extraction Kit.
  5. Measure DNA concentration using Nanodrop 2000.

Double digestion by restriction enzyme
  1. Mix as follows and incubate at 37°C for 2 hours.
    compounds μL
    CutSmart Buffer x10 5
    vector 6
    Enzymes 1 each
    DW X
  2. Electrophorese with the uncut vector to confirm that it was correctly cleaved (only one band in the middle of the two bands of the uncut vector), and then cut the gel out and extract the gel.

Double digestion by restriction enzyme
  1. Mix as follows and incubate at 37°C for 2 hours.
    compounds μL
    CutSmart Buffer x10 5
    vector 6
    Enzymes 1 each
    DW X
  2. Electrophorese with the uncut vector to confirm that it was correctly cleaved (only one band in the middle of the two bands of the uncut vector), and then cut the gel out and extract the gel.

Gibson Assembly
  1. Mix as follows and incubate at 50°C for 30 minutes.
    compounds μL
    Master Mix 3
    Vector 1
    inserts 2 each

Transformation
  1. Place competent cells on ice for 15 minutes to thaw in a microtube. During this time, set the heat block to 56°C.
  2. Add all of the Gibson Assembly reaction solution to the competent cells.
  3. Conduct heat shock placing the cells. 0°C for 5 min, 56°C for 35 sec, and 0°C for 5 min.
  4. Add 100 μL of antibiotic-free liquid LB medium, and recover at 37°C for 1 hour.
  5. Spread the cells on antibiotic-added LB agar medium and incubate at 37°C overnight.

Plasmid extraction
  1. Poke One colony on plate with a sterilized pipette tip.
  2. Put the tip into antibiotic-added liquid LB medium and incubate with shaking until the medium became turbid.
  3. Extract plasmids according to FastGene Plasmid Mini.
  4. Measure DNA concentration using Nanodrop 2000.

Culturing, Compounds Analysis

Pre-culture and main culture
  1. Add one drop of the inoculum to a 15 mL test tube containing antibiotic-added liquid LB medium and incubate at 37°C with shaking.
  2. When the culture medium became turbid, transfer it to a baffled Erlenmeyer flask containing 100 mL of antibiotic-added liquid LB medium and incubate at 37°C, 120 rpm for 3 hours with shaking.

Substance Synthesis
  1. Centrifuge the culture medium and concentrate the bacteria to make a pellet. Collect the supernatant in an appropriate container and autoclaved to sterilize.
  2. Add saline solution to the pellet, vortex to suspend it, and measure OD.
  3. Add saline so that the OD was 10.
  4. To a 50 mL Falcon, add 4.5 mL of medium for material synthesis and 0.5 mL of E. coli suspension. After that, shake for the specified time.

Solvent extraction and LC/MS of CDP
  1. To 3 mL of the reaction solution, add 3 mL of ethyl acetate, shake well, and centrifuge at 10,000 rpm for 1 minute.
  2. Reserve 2.5 mL of the oil layer, add 2.5 mL of ethyl acetate to the remainder, and repeat the same mixing, separation, and preparative procedure twice.
  3. Evaporate 7.5 mL of ethyl acetate solution of the sample in a centrifugal evaporator (Sakuma Seisakusho EC100) at reduced pressure.
  4. Dissolved the remaining solid in 100 μL of methanol, transfer it to a micro tube, and centrifuge at 15,000 rpm for 10 min. collect 40 μL of the supernatant carefully and place in an ampoule to make a sample.
  5. Conduct LC/MS analysis (SHIMADZU LCMS-8045) using the following settings
    Organic solvent: acetonitrile
    Inorganic solvent: formic acid
    time: 26 minutes

Culture Recipe

LB
compounds g
Bacto Tryptone 2
Bacto Yeast Extract 1
NaCl 2
DW up to 200 mL

M9
compounds g
Na2HPO4 0.72
KH2PO4 0.6
NH4Cl 0.2
NaCl 0.1
DW up to 100 mL

PBS
compounds g
Na2HPO4 0.04
KH2PO4 0.01
NaCl 0.9
DW up to 100 mL