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

    Methodology

    Prepare the TAE buffer joining the materials in a volumetric flask, beaker or container at the stated order.

    Add the agarose and TAE in Erlenmeyer. Mix and heat it carefully in the microwave until homogeneous and without crystals.

    Wait the glass cooling until the temperature reaches ~ 60 ° C. Add Syber Safe and pour the solution in the appropriate gel-forming container. Insert the comb to form the wells and wait for gel solidification.

    Remove the comb, place the gel on the running cube, and immerse gel in TAE.

    In a microtube or paraplast tape, mix the loading buffer with the DNA and carefully apply it to the gel. Apply the 1kb DNA Ladder to gel as well.

    Plug the electrodes and run gel for ~40min.

    Glycerol Clone Storage

      Materials used:

    • 50% Glycerol
    • Sterilized tips
    • Sterilized microtubes
    • Pipettes
    • Bacteria in liquid medium containing DNA clone

    Methods

    Homogenize bacteria medium by inversion.

    Near the flame, transfer 170µL of bacterial culture to a sterile microtube.

    Add 30µl of 50% glycerol and homogenize.

    Place the glycerol clones in a -20°C freezer for ~ 20h and then transfer them to a - 80 °C freezer.

    Ligation

    We used the IFN-beta DNA amplified by PCR for ligation with pGEM Vector. For that, Promega’s pGEM-T Vector System I was used

    Reaction - total 10 ul

    2x Rapid Ligation Buffer, T4 DNA Ligase 5 ul
    pGEM-T Vector (50 ng) 1 ul
    IFN-beta PCR product from tube 1 3 ul
    T4 DNA Ligase (3 Weiss units/ul) 1 ul

    Incubation for 1 hour at room temperature. After, the ligation was incubated at 4°C, overnight.

    Pre-inocolum and inocolum

    Materials used:

    Sterilized tips

      Materials used:

    • Pipettes
    • Antibiotic
    • Petri dishes containing isolated bacterial colonies
    • LB Liquid medium
    • Bunsen burner