Team:UFMG Brazil/Protocols

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PCR for IFN-β amplification

~~PCR reaction~~

3 μl incubation buffer
3 μl primer F
3 μl primer R
1 μl INF-β plasmidial DNA (“pUCIDT gene 1-LAP-INFB”)
3 μl dNTP (10x diluted)
17 μl H2O
0.5 μl Taq DNA Polymerase

~~Thermocycler program:~~

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 – 1L~~

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:
8 µL nuclease-free water
2 µL Miniprep DNA
2 µL 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 1 kb DNA Ladder to gel as well.
Plug the electrodes and run gel for ~40 min.

Double digestion

~~Double digestion reaction:~~

Nuclease-free water-4.8 μl
pLP-neo miniprep DNA--12 μl
BSA------0.2 μl
Multicore 10x buffer--2 μl
XbaI enzyme-----0.5 μl
BamHI enzyme----0.5 μl

Final volume: 20 μl
Quick spin
Incubation at 37º C for 3 h
Inactivation at 56º C for 30 min

Ligation

We used the IFN-β DNA amplified by PCR for ligation with pGEM-T Easy vector System I (Promega).

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~~Reaction - total 10 μl~~

2x Rapid Ligation Buffer, T4 DNA Ligase 5 μl
pGEM-T Easy vector (50 ng) 1 μl
IFN-β PCR product from tube 1 3 μl
T4 DNA Ligase (3 Weiss units/μl) 1 μl

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

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) Agar 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 2 g 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 o MgCl2 MW: 95.211 g/mol o 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 o KCl MW: 74.5513 g/mol o 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 TOP10 E.coli bacteria chemo competent transformation 2 μl of pUCIDT gene 1-LAP-INFβ plasmid in 100 μl of TOP10 bacteria 30 min ice incubation 90 s incubation in 42º 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 Bacterial plating Materials used Erlenmeyer flask and sterilized becker LB Agar Sterile petri dishes Antibiotic Bunsen burner 70% Ethanol Platinum strap Clones Methods Heat LB Agar in the microwave. Let it cool near the flame. Pour between 15 mL and 30 mL 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 Pre-inoculum and inoculum Materials used · Sterilized tips Pipettes Antibiotic Petri dishes containing isolated bacterial colonies Sterilized test-tubes LB Liquid medium Bunsen burner 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 20 ml with liquid LB medium Repeat the incubation as above and remove the inserted plasmid through miniprep or maxiprep Glycerol Clone Storage Materials used 50% Glycerol Sterilized microtubes Sterilized tips Pipettes Bacteria in liquid medium containing DNA clone Methods 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.