Team:UFMG Brazil/Project/Protocols

LB medium preparation

Five grams of tryptone, 2,5 g yeast extract, and 5 g NaCl were dissolved in 500 mL deionized water, its pH corrected to 7, 00, and autoclaved on liquid cycle (15psi) for 20 min. The solutions were let cooled and the required antibiotics were added as follows, and kept at 4°C:

LB-Agar medium preparation

Antibiotic free LB medium components were mixed with 7.5g of agar to final volume of 500 mL, dissolved with deionized water, autoclaved (15psi) for 20 min, and before solidification, the respective antibiotic were added at the final concentration specified in Table I, and distributed in Petri dishes. The dishes were kept at 4°C.

Preparation of competent cells

A single clone of E. coli XLI blue and DH5α were inoculated into 50 mL LB medium, kept growing overnight at 37°C with 250 rpm shaking. Then, 4 mL of the culture were transferred to 400 mL LB medium and grow approximately 8 hours (OD600 = 0,390 XLI Blue and 0,4 DH5α).

The culture was aliquoted in pre-chilled 50 mL tubes and maintained on ice for 10 min. Then, tubes were centrifuged for 7 min, 1600 g at 4°C and supernatants poured off. Pellets were resuspended with 10 mL ice-cold CaCl2 with the tubes kept on ice. A second centrifugation steps was done for 5 min (1100 g, 4°C). The supernatants were poured off and the cells resuspended with 10 mL ice-cold CaCl2, once again tubes were kept on ice and maintained there for 30 min.

The last centrifugation step was repeated and after supernatants discarded, the cells resuspended with 2 mL of ice-cold CaCl2, dispensed into chilled sterile polypropylene tube in aliquot of 50 µL, and immediately freeze at -80°C. Cells competency were analyzed with iGEM Bba_J23119 plasmid.

Cells Transformation

The competent cell 0.6 tube were thawed in hand, kept on ice and added 300pg of DNA from iGEM distribution kit required parts and maintained on ice for 10min. In water bath at 42°C cells were heat shocked for 2 min, and immediately after, 500 µL of pre-heated LB medium were added to the tube and then transfer to another 1,5 mL tube filled with 500 µL pre-heated LB medium, and incubated at 37°C, 250rpm for one hour.

The LB-agar plates were heated at 37°C in between culture incubation time. After the initial incubation, 40 µL were transferred to the plate and, with an iron loop, the content were distributed on top of the agar, and incubated for 16 hours.

One colony of each plate was selected and transfer to a 50 mL LB medium and growth overnight. Then, plasmid purification was performed.

Plasmid Purification

Quiagen purification plasmids were used to perform this step as manufacture instruction. Briefly, culture cells were transfer to a 5 mL tube and centrifuged at 6.800 g for 3 min at room temperature. Pelleted cells were resuspended in 250 µL buffer P1 and transfer to a 1,5 mL microcentrifuge tube, and then added 250 µL of P2 buffer and gently mixed by tube inverting. To neutralize the lyses buffer, 350 µL of N3 buffer were added and mixed. The solutions were then centrifuged for 10 min at 17.900 g. This step supernatant were applied into a QIAprep spin column, and centrifuged for 60 seconds. Wash buffer PB and PE were respectively (0.5 and 0.75 mL) added to the column and spun for 30s. One final spin were done for one minute to remove wash buffer, and the plasmid eluted with 50 µL of EB buffer by centrifugation. To assess the plasmids yield we use The Thermo Scientific™ NanoDrop Lite Spectrophotometer.

Amplification

Designed genes were amplified by standard PCR, using primes design for RFC25-Preffix and RFC25-Suffix (5´- CGT AGG AAT TCG CGG CCG CTT CTA GAT GGC CGG C – 3´ and 5´- TTC GTC TGC AGC GGC CGC TAC TAG TAT TAA CCG GT – 3´).

Amplifications were performed in a M.J. Research PTC-100 (GMI Inc.) thermocycler. The amplification program was designed as follows:Initial denaturation: 10 minutes at 94ºC. 30 cycles of denaturation (94ºC for 1 minute), annealing (55ºC for 1 minute) and extension (72ºC for 1 minute). The final extension was performed at 72ºC.- PCR products were analyzed in 1% agarose gels, and 1Kb DNA Ladder (Invitrogen) was used.
Gels were stained with ethidium bromide, and photographed using a U.V. table transilluminator connected to a CPU.

Long DNA purification

To validate the long DNA presence in cancer, different mice with induced colon cancer model had its feces collected, kept in -80ºC until its DNA purification. The purification was made using TRIzol® Reagent (Life Technology) protocol. Briefly, frozen feces 500uL of TRIzol®, and homogenized; 0.2 mL of chloroform was added, shacked, and centrifuged for 15 minutes (12,000 g) at 4°C. The interphase was kept, mixed with 0.15 mL of 100% ethanol, and centrifuged at 2000 g for 5 minutes at 4°C. The DNA pellet was washed with 0.5 mL 0.1M sodium citrate in 10% ethanol, incubated for 30 minutes, and centrifuged for 5 minutes at 2,000 g (4°C) two times. Ethanol 75% (1 mL) was added, incubated for 20 minutes, and centrifuged at 2,000 g. The DNA was resuspended in 8mM NaOH (0.2 µg/mL), and corrected with HEPES (pH=7.00).

Assembly Workflow