Miniprep - Kit-free high throughput protocol - source code

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Revision as of 05:49, 19 November 2009 by Vaishnavi Ananth (Talk | contribs)
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#include "BioCoder.h"

void main()
{

	start_protocol("Miniprep - Kit-free high-throughput protocol");

	Fluid culture = new_fluid("overnight culture containing your plasmid");
	Fluid stet = new_fluid("STET buffer", "8% sucrose, 50 mM Tris-HCl (pH 8), 0.5% Triton X-100, 50 mM EDTA");
	Fluid lysozyme = new_fluid("lysozyme (10mg/ml)");
	Fluid isoprop = new_fluid("isopropanol",  ICE_COLD);
	Fluid eth80 = new_fluid("80% ethanol", ICE_COLD);
	Fluid te = new_fluid("TE buffer", "10 mM Tris-HCl (pH 8), 1 mM EDTA ");

	Container flask = new_container(FLASK);
	Container eppendorf1 = new_container(EPPENDORF);

	// 1. Transfer 1.5 mL of an overnight culture containing your plasmid to an eppendorf tube and spin at 5000 rpm for 5 min in a tabletop centrifuge to pellet the cells.
	// 2. Remove and discard the supernatent.
	first_step();
	set_container(culture, flask);
	measure_fluid(flask, vol(1.5, ML), eppendorf1);
	centrifuge_pellet(eppendorf1, speed(5000, RPM), RT, time(5, MINS));

	// 3. Add 300 μL STET buffer, and resuspend cells by vortexing.
	next_step();
	measure_and_add(eppendorf1, stet, vol(300, UL));
	resuspend(eppendorf1);
	
	// 4. Add 10 μL lysozyme (10 mg/mL), vortex, and submerse in boiling water for 40 sec.
	next_step();
	measure_and_add(eppendorf1, lysozyme, vol(10, UL));
	vortex(eppendorf1);
	store_for(eppendorf1, 100, time(40, SECS));

	// 5. Spin for 30 min in a tabletop centrifuge at maximum speed at 4 ˚C.
	next_step();
	centrifuge_pellet(eppendorf1, speed(SPEED_MAX, RPM), 4, time(30, MINS));

	// 6. Remove pellet from each tube with a toothpick. The cellular debris should stick well to the toothpick. Try to insert and remove the toothpick from the center of the tube so you don't get any cellular debris on the sides of the tube.
	next_step();
	comment("Remove pellet from each tube with a toothpick. The cellular debris should stick well to the toothpick. Try to insert and remove the toothpick from the center of the tube so you don't get any cellular debris on the sides of the tube.");

	// 7. Add 300 μL ice cold isopropanol to precipitate the DNA (or 300μL of 2:1 isopropanol:ammonium acetate, mixed just before you use it. See this discussion of precipitating nucleic acids.)
	next_step();
	measure_and_add(eppendorf1, isoprop, vol(300, UL));
	comment("This is done to precipitate the DNA.");

	// 8. Spin for 10 min in a tabletop centrifuge at maximum speed at 4 ˚C.
	// 9. Remove supernatent.
	next_step();
	centrifuge_pellet(eppendorf1, speed(SPEED_MAX, RPM), 4, time(10, MINS));

	//10. Add 200 μL ice cold 80% ethanol to wash pellet and spin for 5 min in a tabletop centrifuge at maximum speed at 4 ˚C.
	//11. Remove supernatent.
	next_step();
	measure_and_add(eppendorf1, eth80, vol(200, UL));
	comment("This is to wash the pellet.");
	centrifuge_pellet(eppendorf1, speed(SPEED_MAX, RPM), 4, time(5, MINS));

	//12. Dry pellet (air dry at room temperature or 37 ˚C or dry in a speedvac).
	next_step();
	begin_option();
	dry_pellet(eppendorf1, "in air");
	next_option();
	dry_pellet(eppendorf1,"in a speedvac");
	end_option();

	//13. Rehydrate in 50 μL TE. 
	next_step();
	measure_and_add(eppendorf1, te, vol(50, UL));
	resuspend(eppendorf1);

	end_protocol();
}
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