Team:UFMG Brazil/Protocols
Project
Lab Work
Modeling
Practices
Synenergene
Team
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
- 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% Alcohol
- 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.
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
- 50% Glycerol
- Sterilized tips
- Sterilized microtubes
- Pipettes
- Bacteria in liquid medium containing DNA clone
Materials used:
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
- Pipettes
- Antibiotic
- Petri dishes containing isolated bacterial colonies
- LB Liquid medium
- Bunsen burner
Materials used:
Methodology
- 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 20ml with liquid LB medium
- Repeat the incubation as above and remove the inserted plasmid through miniprep or maxiprep
Materials used:
TOP10 E.coli bacteria chemo competent transformation
2 ul of pUCIDT gene 1-LAP-INFB plasmid in 100 ul of TOP10 bacteria 30 min ice incubation 90s incubation in 42ºC water bath;
2 min ice incubation; Carefully add 0.9 mL of SOC medium; 1h incubation in 37 °C and 120 rpm of rotation;
2 bacterial plating of 100 ul of transformation (37°C, 16h) Each plate was made with 30 ml of LB agar and 30 ul of ampicillin 100 mg/ml
