Team:UFMG Brazil/Protocols




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Protocols

Bacteria mediums

*LB (Luria-Bertani) liquid medium – 300 ml
3 g tryptone
1.5 g yeast extract
3 g NaCl
Complete to 300 ml with distilled water
Autoclave in a 500 ml erlenmeyer
Prepare 2 erlenmeyers with 300 ml of medium each

*LB (Luria-Bertani) liquid medium – 300 ml
3 g tryptone
1.5 g yeast extract
3 g NaCl
4.5 g Agar (weigh directly into the erlenmeyer)
Complete to 300 ml with distilled water
Autoclave in a 500 ml erlenmeyer

*SOC medium – 100 ml 2g tryptone
0.5 g yeast extract
200 µl NaCl 5 M
1 ml MgCl2 1 M
250 µl KCl 1 M
10 ml MgSO4 1 M
Complete to 100 ml with distilled water
Autoclave and store in bottle with lid

  • Preparing NaCl 5 M
    • NaCl MW: 58.44 g/mol
    • 58.44 g ---- 1 mol ---- 1000 ml
      • x ------------------------- 50 ml x = 2.92 g
    • 2.92 g -------- 1 mol
      • y --------------- 5 mol y = 14.6 g

    Put 14.6 g NaCl into a 50 ml falcon tube and complete to 50 ml with distilled water

  • Preparing MgCl2 1 M
    • MgCl2 MW: 95.211 g/mol
    • 95.21 g ---- 1 mol ---- 1000 ml
      • x ------------------------- 50 ml x = 4.7 g
      Put 4.7 g MgCl2 into a 50 ml falcon tube and complete to 50 ml with distilled water.

  • Preparing KCl 1 M
    • KCl MW: 74.5513 g/mol
    • 74.55 g ---- 1 mol ---- 1000 ml
      • x ------------------------- 50 ml x = 3.72 g
      Put 3.72 g KCl into a 50 ml falcon tube and complete to 50 ml with distilled water

    Bacteria planting

    Materials used


    • Erlenmeyer flask and sterilized becker
    • LB Agar
    • Sterile petri dishes
    • Antibiotic
    • Bunsen burner
    • 70% Alcohol
    • Platinum strap
    • Clones

    Methods

    Heat LB Agar in the microwave. Let it cool near the flame.

    Pour between 15mL and 30mL of LB Agar in an Erlenmeyer. Add the antibiotic in the right proportion and mix

    Pour the medium in the Petri dish and let it solidify near the flame.


    Pipet the desired volume of clone on the plate. Spread the bacterial suspension throughout the petri dish homogeneously.

    Cover the plate and incubate it at 37 °C for 12-16

    Standard antibiotics concentration used:


    • Ampicillin - 10 µl / ml
    • Chloramphenicol -1.2 µL / ​​mL

    Double digestion

    Double Digestion reaction:

    Nuclease-free water 4,8ul
    pLP-neo miniprep DNA 12ul
    BSA 0,2ul
    Multicore 10x buffer 2ul
    XbaI enzyme 0,5ul
    bamHI enzyme 0,5ul

    Final volume: 20ul.

    Quick spin

    Incubation at 37ºC for 3 hours.


    Inactivation at 56ºC for 30 minutes


    Agarose DNA electrophoresis gel

    Gel materials – 30mL gel 1.2%


    • 0.36g agarose
    • 30mL TAE Buffer (tris-acetate-EDTA)
    • 1.5µL Sybr Safe

    TAE Buffer materials – 1L


    • 242g Tris Base
    • 57.1 mL Cold Acetic Acid
    • 100mL 0.5M EDTA
    • 1L distilled water

    Sample materials - 12µL


    • 1kb DNA ladder
    • 10µL DNA samples:
        • 8µL nuclease-free water
        2µL Miniprep DNA
    • 2µL loading buffer