Team:Virginia/Protocols

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Hello World

QIAGEN Mini-Prep

  • Spin cell broth down in centrifuge at >13200 revolutions per minute in microcentrifuge (generally 4 minutes is adequate).
  • Pour off remaining broth and resuspend in 250 uL of Buffer P1 (LyseBlue is nice to have).
  • Add 250 uL Buffer P2 (for cell lysis). Invert microcentrifuge tubes 4-6 times. Do NOT allow to run for more than 5 minutes.
  • Add 350 uL Buffer N3 and invert tubes another 4-6 times.
  • Spin in microcentrifuge at >13000 revolutions per minute for ten minutes.
  • Transfer supernatant to provided spin column.
  • Run spin column in microcentrifuge for 60 seconds. Discard flow-through.
  • Add 0.5 mL Buffer PB. Centrifuge for 60 seconds. Discard flow-through.
  • Add 0.75 mL Buffer PE. Centrifuge for 60 seconds. Discard flow through.
  • Centrifuge for an additional 60 seconds.
  • Transfer spin-column top to a microcentrifuge tube for collection.
  • Add 50 uL Buffer EB to center of column. Allow to sit for one minute.
  • Centrifuge for one minute. The clear liquid in the bottom is your DNA solution.


Electrophoresis Gel (Making)

  • In a 100 mL Erlenmeyer flask, place and pour: 0.5 g Agar and 50 mL 1X TAE Buffer
  • Microwave for 30 seconds and then swirl the flask
  • Microwave for 15 seconds (caution: do not let the solution boil out of the flask)
  • Pipette 2 microliters of Ethidium Bromide into the gel solution and then swirl flask
    • Ethidium Bromide stains the plate for DNA to be observed under UV light
  • NOTE: Ethidium Bromide is a hazardous chemical and must be disposed of properly - this includes the gel itself
  • Setup gel holder with desired size and number comb perpendicular leads so that liquid gel stays contained
  • Pour all of hot gel into gel holder, cover with saran wrap and leave out to solidify


Gel Electrophoresis (Loading & Running)

  • Align wells on the side of the negetive(black) lead
  • Fill with 1X TAE buffer to cover gel with thin layer of buffer
  • carefully remove the comb
  • Using small epindorf tubes mix together DNA and 6X loading dye with a 6:1 ratio (this ratio is dependant on the loading dye)
  • Mix 1 uL of 1kb ladder with 5 uL of water and 1 uL of 6X loading maintaining the ratio
  • Carefully pipette the DNA mixtures into the wells being careful not to pierce the bottom of the wells
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