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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.

PCR, electrophoresis, and gel extraction

  1. Mix as follows. Modify the values according to the concentrations of the DNA or the template.
  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.
  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.
compounds μL
データセル1-2 データセル1-2
データセル2-1 データセル2-2
データセル2-1 データセル2-2
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