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 mini prep

  • P1: 1 molar 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.

    • Plasmid Mini prep by alkaline lysis

    1. Take 15 ml over night culture, centrifuge at max speed 10 minutes
    2. Remove the supernatant and re-suspend the Pellet in 2 ml resuspension buffer
    3. Add 4 ml lysis buffer, mix by invesion
    4. Incubate 5 mins at RT, add 3ml neutralization buffer, incubate 15 mins on ice.
    5. Centrifuge at max speed for 10 mins "Cooling centrifuge 1000g"
    6. Take about 8 ml supernatant and add 0.6vol isopropanol
    7. Centrifuge at Max speed for 10 mins then remove the supernatant
    8. Wash the pellet using 70% ethanol, discard the ethanol and then air dry
    9. Add 200ul TE buffer to the pellet "Resuspend and transfer to new epp"
    10. Add another 200ul TE buffer to the new eppendorf
    11. Add 10ul RNAse then incubate 15 mins at 37C
    12. Add equal volume of Phenol chloroform , vortex, centrifuge at max speed 2 mins
    13. Transfer the upper layer to new epp then add equal volume of chloroform, vortex, centrifuge at max speed 2 mins
    14. Transfer the upper layer to new eppendorf the add 1/10 vol 3M Na.acetate then add 2.5vol absolute ethanol.
    15. Centrifuge 15 mins at 4C
    16. Discard supernatant, Wash the pellet using 70% ethanol, remove the ethanol then spin down
    17. Re-suspend the pellet in TE buffer
    18. Measure the concentration and purity