DNA extraction - Salting Out - source code
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<code> #include "BioStream.h" void main() { start_protocol("DNA Extraction - salting out"); Fluid dig_buffer = new_fluid("Digestion Buffer","10mM NaCl, 10mM TRIS (pH 8.0), 10mM EDTA (pH 8.0), 0.5% SDS"); Fluid proteinasek = new_fluid("Proteinase K", "20mg/ml"); Fluid naac = new_fluid("Sodium Acetate pH 5.2", "3M"); Fluid eth98 = new_fluid("98% ethanol", ICE_COLD); Fluid eth70 = new_fluid("70% ethanol", ICE_COLD); Fluid te = new_fluid("1X TE"); Fluid water = new_fluid("water"); Tissue req_tissue = new_solid("tissue"); Container sterile_microfuge_tube1 = new_container(STERILE_MICROFUGE_TUBE); Container sterile_microfuge_tube2 = new_container(STERILE_MICROFUGE_TUBE); Container sterile_microfuge_tube3 = new_container(STERILE_MICROFUGE_TUBE); Container sterile_microfuge_tube4 = new_container(STERILE_MICROFUGE_TUBE); // * Tissue Digestion first_step("Tissue Digestion"); first_sub_step(); set_container(req_tissue, sterile_microfuge_tube1); //1. Add 5μL Proteinase K to each mL of Digestion Buffer (final 0.5mg/mL) measure_fluid(dig_buffer, sterile_microfuge_tube2); measure_prop_and_add(sterile_microfuge_tube2, proteinasek, 0.005); comment("That is, for each ml of Digestion Buffer, add 5 µl of ProteinaseK."); name_sample(sterile_microfuge_tube2, "solution"); //2. Homogenise (or simply place) tissue in solution next_sub_step(); homogenize_tissue(sterile_microfuge_tube1, sterile_microfuge_tube2.contents); //3. Incubate at 55°C for 1 hour to overnight next_sub_step(); incubate(sterile_microfuge_tube2, 55, time_range(1, 12, HRS)); //4. Mix by vortexing then centrifuge at maximum speed in a benchtop centrifuge for 2 minutes next_sub_step(); vortex(sterile_microfuge_tube2); centrifuge_phases(sterile_microfuge_tube2, speed(SPEED_MAX, RPM), 4, time(2, MINS), sterile_microfuge_tube3); //5. Transfer supernatant into a new tube // * Precipitation of Protein and Cell Debris next_step("Precipitation of Protein and Cell Debris"); //1. Add 1/10 volume of Sodium Acetate 3M pH 5.2 (final 0.3M) first_sub_step(); measure_prop_and_add(sterile_microfuge_tube3, naac, 0.1); //2. Invert to mix and incubate at -20°C for ~15 minutes next_sub_step(); invert(sterile_microfuge_tube3); incubate(sterile_microfuge_tube3, -20, time(15, MINS)); //3. Centrifuge (preferably at 4°C) at maximum speed in a benchtop centrifuge for 20 minutes centrifuge_phases(sterile_microfuge_tube3, speed(SPEED_MAX, RPM), 4, time(20, MINS), sterile_microfuge_tube4); comment("Be careful not to transfer any of the white solid (cell debris and SDS) into the fresh tube."); //4. Transfer supernatant to a new tube // * Precipitation of Nucleic Acids next_step("Precipitation of Nucleic Acids"); //1. Add ~2 volumes of 98% ethanol (final 60-80%) first_sub_step(); measure_prop_and_add(sterile_microfuge_tube4, eth98, 2); //2. Invert to mix and incubate at -20°C for ~15 minutes next_sub_step(); invert(sterile_microfuge_tube4); incubate(sterile_microfuge_tube4, -20, time(15, MINS)); //3. Centrifuge (preferably at 4°C) at maximum speed in a benchtop centrifuge for 20 minutes next_sub_step(); centrifuge_pellet(sterile_microfuge_tube4, speed(SPEED_MAX, RPM), 4, time(20, MINS)); //4. Wash pellet with 98% ethanol, and once or twice with 70%. Allow to air dry then resuspend in water or 1xTE next_sub_step(); measure_and_add(sterile_microfuge_tube4, eth98, vol(1, ML)); vortex(sterile_microfuge_tube4); centrifuge_pellet(sterile_microfuge_tube4, speed(SPEED_MAX, RPM), 4, time(5, MINS)); measure_and_add(sterile_microfuge_tube4, eth70, vol(1, ML)); vortex(sterile_microfuge_tube4); centrifuge_pellet(sterile_microfuge_tube4, speed(SPEED_MAX, RPM), 4, time(5, MINS)); optional_step(); measure_and_add(sterile_microfuge_tube4, eth70, vol(1, ML)); vortex(sterile_microfuge_tube4); centrifuge_pellet(sterile_microfuge_tube4, speed(SPEED_MAX, RPM), 4, time(5, MINS)); next_step(); dry_pellet(sterile_microfuge_tube4, "in air"); begin_option(); measure_and_add(sterile_microfuge_tube4, te, vol(10, UL)); next_option(); measure_and_add(sterile_microfuge_tube4, water, vol(10, UL)); end_option(); resuspend(sterile_microfuge_tube4); comment("Ensure to dry the pelletted DNA completely before attempting to resuspend."); end_protocol(); } </code>