Team:Cairo Egypt/Experiments

Experiments and Protocols

General Preparation

Gel Preparation

  1. Prepare 50 mL of 0.7% agarose gel. Add 0.35 g of agarose to 50 mL of TAE buffer and boil in microwave until all agarose is melted. Stop microwave every 20 to 30 seconds and swirl the solution to help the agarose dissolve. Required about 2 minutes for all of the agarose to dissolve. a. 50mL 1X TAE buffer i. 10 mL of 50X TAE ii. 490 mL of DI water
  2. Let agarose gel solution cool to about 55C (hot but not burning to the touch). This will take only a few minutes. Take gel solution to the Gulari lab and add 10 uL of ethidium bromide (10 mg/mL). Swirl solution to mix for at least 30 seconds
  3. Get about a 9 cm gel cast and put barriers at each end (make sure they are snug). Pour gel solution into cast, try not to create bubbles/foam.
  4. Use pipette tip to pop any bubbles. If bubbles will not pop, use tip to move the to the sides of the gel.
  5. Put comp into gel to create wells. Use the 13x comb. Put the comb near the beginning of the gel, second notch from the rubber stop. Leave gel on bench 20-25 minutes to solidify.

Preparation of Antibiotics

  1. Ampicillin 100 µg/mL
  2. Tetracycline 10 µg/mL
  3. Chloramphenicol 25 µg/mL
  4. Kanamycin 50 µg/mL

Preparation of LB media

  1. Dissolve 10 g tryptone, 5 g yeast extract, and 10 g NaCl in 950 mL deionized water.
  2. Adjust the pH of the medium to 7.0 using 1N NaOH and bring volume up to 1 liter.
  3. Autoclave on liquid cycle for 20 min at 15 psi. Allow solution to cool to 55°C, and add antibiotic if needed (50µg / mL of Amp or Kan).
  4. Store at room temperature or +4°C.

Preparation of LB-Agar

  1. Prepare LB medium as above, but add 15 g/L agar before autoclaving.
  2. After autoclaving, cool to approx. 55°C, add antibiotic (if needed), and pour into petridishes.
  3. Let harden, then invert and store at +4°C in the dark

Protocol To make 500mL of LB agar (makes about 25 LB agar plates):

Weigh out the following into a 1L Erlenmeyer flask:

  • 5g NaCl
  • 5g Tryptone
  • 2.5g Yeast Extract
  • 7.5g Agar
  • add dH2O to 500mL

Preparation of reagents for manual plasmid miniprep

  • P1: 1molar Tris PH 8 5ml , .5 molar EDTA 2ml ,glucose , Distilled water up to 100ul
  • P2: .2 molar NAOH 1% SDS
  • P3: 5 molar sodium acetate (adjusted with glacial acetic acid ) or 2.55 molar potassium acetate.