Difference between revisions of "Team:UFMG Brazil/Protocols"

• 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

• 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.

• 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% Ethanol
• Platinum strap
• Clones

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 h.

Standard antibiotics concentration used:

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

Double digestion

Double Digestion reaction:

Final volume: 20 µl.

Quick spin

Incubation at 37° C for 3 h.

Inactivation at 56° C for 30 min.

Agarose DNA electrophoresis gel

Gel materials – 30 mL gel 1.2%

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

TAE Buffer materials – 1 L

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

Sample materials - 12 µL

• 1 kb DNA ladder
• 10 µL 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 ~40 min.

Glycerol Clone Storage

Materials used:

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

Homogenize bacteria medium by inversion.

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

Add 30 µl of 50% glycerol and homogenize.

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

Ligation

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

Reaction - total 10 µl

Incubation for 1 h 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

Methodology

Materials used:

• Add 4 ml of LB liquid medium to each test-tube and the correspondent antibiotic ratio
• With toothpick or sterilized tip, remove an isolated colony
• Place the toothpick / tip inside the test-tube, without touching it sides
• Leave the tubes overnight at 37° C for 16 h in the shaker
• After incubation, remove the tubes from the shaker. Medium must be turbid, indicating bacterial growth
• Pour the contents of the test-tubes on a larger recipient and complete volume to 20 ml with liquid LB medium
• Repeat the incubation as above and remove the inserted plasmid through miniprep or maxiprep

TOP10 E.coli bacteria chemo competent transformation

2 µl of pUCIDT gene 1-LAP-IFN-β plasmid in 100 µl of TOP10 bacteria
30 min ice incubation
90 s incubation in 42° C water bath;

2 min ice incubation;
Carefully add 0.9 mL of SOC medium;
1 h incubation in 37° C and 120 rpm of rotation;

2 bacterial plating of 100 µl of transformation (37° C, 16 h)
Each plate was made with 30 ml of LB Agar and 30 µl of ampicillin 100 mg/ml