Team:GenetiX Tec CCM/Experiments
Protocol 1
Preparation of culture media (1L). 1.- 5 g of yeast 2.- 10 g of sodium chloride. 3.- 10 g of triptone. 4.- Add water till 1 uL volume.
Protocol 2
Competent cell´s preparation 1.- Centrifug your media culture tube (25 mL) at 4,000 rpm during 15 min. 2.- Pour the supernatant and add 10 mL of cold MgCl2 solution. Centrifuge them at 4,000 rpm at 4 °C during 15 min. 3.- Pour the supernatant and resuspend the pellet in 40 mL of cold CaCl2 solution. Keep it in ice during 20 min and place them in eppendorf tubes. Centrifuge them at 4,000 rpm at 4°C during 15 min. 4.- Pour the supernatant and resuspend the pellet in 40 mL of cold 85mM CaCl2/15% glycerlol v/v solution. Centrifuge again at 2,100 rpm at 4°C during 15 min. 5.- Pour the supernatant and resuspend in 2 mL of cold 85bmM CaCl2/15% glycerol v/v solution. 6.- Aliquote in tubes of 100 uL previously frozen and store at -80°C.
Protocol 3
DNA extraction from Buffy coat: 1.- Centrifuge 5 mL of culture medium at 1,500 rpm for 10 min at 4°C. 2.- Carefully transfer 600 uL of buffy coat to a microtube. 3.- Add 1 mL of RBC buffer and mix throughly by vortex. 4.- Centrifuge at 4,500 rpm during 30 s and pour carefully so your pellet is not wasted. 5.- Repeat steps 2 and 3 adding just 500 uL from RBC buffer. 6.- Once separated, add 300 uL of lysis buffer to the pellet and mix by vortex. 7.- Add 150 uL of protein precipitation solution. 8.- Centrifuge at 11,000 rpm during 5 min; then, transfer the supernatant to a new microtube. 9.- Add 700 uL of cold isopropanol and mix by inverting until you see a white formation within the tube. 10.- Centrifuge at 11,000 rpm during 4 min and pour carefully taking care of the pellet at the bottom. 11.- Add 500 uL of cold ethanol 70% to wash and then centrifuge at 11,000 rpm during 4 min. 12.- Pour and let the ethanol evaporate at room temperature. 13.- Resuspend the DNA pellet with 12 uL of TE buffer; incubate all night at 4 °C.
Protocol 4
Electrophoresis: 1.- Prepare 1L of TAE 10X (48.4g of Tris base[ tris (hidroxymethyl) amminomethane], 11.4 mL of glacial acetic acid (17.4 M) and 3.7 g of EDTA then fill with deionized water to 1 L. 2.- Prepare a 50 mL solution of 3% agarose using TAE buffer (without using water). 3.- Heat the gel in the microwave in intervals of 30 s, 20 s and 10 s until the agarose is completely dissolved, taking care that it does not boil so much. 4.- Let cool down the gel (55-60°C) and pour in a base with the comb already on its place to form the charging wells. 5.- Wait until the gel solidifies. 6.- Remove the comb and place the base with the gel inside the electrophoretic chamber. 7.- Add TAE buffer until the gel is covered. 8.- Take 5 uL of each sample from the PCR and 1 uL of charging gel in a new PCR tube, mix well. 9.- Charge the samples inside the wells of the gel, adding a weight marcker. 10.- Close the electrophoretic chamber and run at 120 V during 30 min. Follow the displacement ofthe samples. 11.- Take out the gel from the chamber very carefully and place in the UV light to see the DNA strands.
Protocol 5
Competent cell transformation: 1.- Place 1 uL of the plasmid in a microtube, gently add 100 uL of your competent cells. Take another tube and place only 100 uL of competent cells, so you have a control group. 2.- Mix gently with a pipette and incubate during 30 min in ice. 3.- Ab ruptly place your cells in 42°C dry bath during 2 min and replace in ice whwn finished. 4.- Add 900 uL of LB medium and incubate at 37 °C for 30 min. 5.- Inoculate with 200 uL of your transformed cells in LB-Amp. 6.- Incubate overnight and store at 4 °C of freeze in 15-25% glycerol.