Kafatos:Minipreps protocol - source code

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Revision as of 05:48, 19 November 2009 by Vaishnavi Ananth (Talk | contribs)
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#include "BioCoder.h"

void main()
{
	start_protocol("Katafos - Minipreps");

	Fluid lb = new_fluid("5 ml sterile LB medium (+ antibiotic)");
	Fluid p1 = new_fluid("P1 Buffer");
	Fluid p2 = new_fluid("P2 Buffer");
	Fluid p3 = new_fluid("P3 Buffer", ICE_COLD);
	Fluid etoh = new_fluid("absolute EtOH");
	Fluid etoh70 = new_fluid("70% ethanol");
	Fluid te = new_fluid("TE");
	Fluid water = new_fluid("ddH<sub>2</sub>O");
	Solid colony = new_solid("single bacterial colony");

	Container flask = new_container(FLASK);
	Container eppendorf1 = new_container(EPPENDORF);
	Container eppendorf2 = new_container(EPPENDORF);

	//Day 1
	//
	//- inoculate 5 ml sterile LB medium (+ antibiotic ?) with single bacterial colony
	//- grow ON @ 37˚C while shaking vigorously
	first_step("DAY 1");
	set_container(lb, flask);
	inoculation(flask, colony, 37, time(12, HRS), 1);

	//[edit] Day 2
	//
	//- spin 1.5 ml 2’ @ 8,000 rpm in table top centrifuge
	next_step("DAY 2");
	first_sub_step();
	measure_fluid(flask, vol(1.5, ML), eppendorf1);
	centrifuge_pellet(eppendorf1, speed(8000, RPM), RT, time(2, MINS));


	//- resuspend pellet in 100 μl P1 Buffer. Let sit 5’ @ RT
	next_sub_step();
	measure_fluid(p1, vol(100, UL), eppendorf1);
	resuspend(eppendorf1);
	store_for(eppendorf1, RT, time(5, MINS));


	//- add 200 μl P2 Buffer, mix and let sit no more than 5’ @ RT
	next_sub_step();
	measure_fluid(p2, vol(200, UL), eppendorf1);
	store_for(eppendorf1, RT, max_time(5, MINS));


	//- add 150 μl ice cold P3 Buffer, mix well and place 5 – 10’ on ice
	next_sub_step();
	measure_fluid(p3, vol(150, UL), eppendorf1);
	vortex(eppendorf1);
	store_for(eppendorf1, ON_ICE, time_range(5, 10, MINS));

	//- spin 10’ @ max rpm (4˚C if possible)
	next_sub_step();
	centrifuge_phases(eppendorf1, speed(SPEED_MAX, RPM), 4, time(10, MINS), eppendorf2);

	//- transfer supernatant to new tube and add 2 volumes of abs EtOH. Let sit for 10 – 20’ on ice
	next_sub_step();
	measure_prop_and_add(eppendorf2, etoh, 2);
	store_for(eppendorf2, ON_ICE, time_range(10, 20, MINS));

	//- spin 10’ @ max. speed @ 4˚C
	next_sub_step();
	centrifuge_pellet(eppendorf2, speed(SPEED_MAX, RPM), 4, time(10, MINS));

	//- discard supernatant and wash pellet with 1ml of 70% EtOH
	next_sub_step();
	measure_fluid(etoh70, vol(1, ML), eppendorf2);
	pipet(eppendorf2);

	//- spin 5’ @ max speed
	next_sub_step();
	centrifuge_pellet(eppendorf2, speed(SPEED_MAX, RPM), RT, time(5, MINS));

	//- suck up or decant supernatant, speedvac or dry pellet @ 37˚C (do not overdo it, though, or it will not properly dissolve again)
	next_step();
	first_option();
	dry_pellet(eppendorf2, "in air");
	next_option();
	dry_pellet(eppendorf2, "in speedvac");
	end_option();
	comment("Do not overdo drying, or the pellet will not properly dissolve again.");

	//- resuspend pellet in 30 μl TE Buffer or ddH20 and check on gel
	next_step();
	first_option();
	measure_fluid(te, vol(30, UL), eppendorf2);
	next_option();
	measure_fluid(water, vol(30, UL), eppendorf2);
	end_option();
	resuspend(eppendorf2);
	electrophoresis(eppendorf2);

	end_protocol();
}
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