Erman's Lab:DNA Miniprep with Alkaline Lysis protocol - source code

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Revision as of 05:53, 19 November 2009 by Vaishnavi Ananth (Talk | contribs)
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#include "BioCoder.h"

void main()
{
	start_protocol("Erman's lab - DNA miniprep with alkaline lysis");

	Fluid lb = new_fluid("2 ml of LB (+ antibiotics)");
	Fluid sln1 = new_fluid("Alkaline Lysis SLN1", "20mM Tris(pH 8), 50mM Glucose, 10mM EDTA");
	Fluid al2 = new_fluid("freshly prepared AL2", "0.2N NaOH, 1% SDS");
	Fluid al3 = new_fluid("AL3", "3M KAc, glacial acetic acid");
	Fluid isoprop = new_fluid("isopropanol", RT);
	Fluid etoh70 = new_fluid("70% EtOH");
	Fluid te = new_fluid("TE");
	Fluid water = new_fluid("water");
	Solid colony = new_solid("single colony");

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

	//1. o/n grow single colony in 2ml of LB (+ antibiotics)
	first_step();
	set_container(lb, flask);
	inoculation(flask, colony, 37, time(12, HRS), 1);
	
	//2. 1.5 ml into eppendorf.
	next_step();
	measure_fluid(flask, vol(2, ML), eppendorf1);

	//3. Pellet cells at max speed in cold room for 1.5 min.
	next_step();
	centrifuge_pellet(eppendorf1, speed(SPEED_MAX, G), 4, time(1.5, MINS));

	//4. Discard supernatant , leave pellet as dry as possible.
	next_step();
	comment("Leave pellet as dry as possible.");

	//5. Resuspend pellet in 100 μL Alkaline Lysis SLN1,vortex.
	next_step();
	measure_fluid(sln1, vol(100, UL), eppendorf1);
	resuspend(eppendorf1);

	//6. Add 200μL freshly prepared AL2, mix by inverting 5-6 times(DO NOT vortex!)
	next_step();
	measure_fluid(al2, vol(200, UL), eppendorf1);
	invert(eppendorf1, 5, 6);
	comment("Do not vortex!");

	//7. Put on ice.
	next_step();
	store(eppendorf1, ON_ICE);

	//8. Add 300μL AL3,invert tubes to mix.
	next_step();
	measure_fluid(al3, vol(300, UL), eppendorf1);
	invert(eppendorf1);

	//9. Incubate on ice for 5 min.
	next_step();
	incubate(eppendorf1, ON_ICE, time(5, MINS));

	//10. Centrifuge at max speed for 5 min in cold room.
	//11. Take the supernatant into a new tube.
	next_step();
	centrifuge_phases(eppendorf1, speed(SPEED_MAX, RPM), 4, time(5, MINS), eppendorf2);
	
	//12. Add 900 isopropanol at RT. Vortex
	next_step();
	measure_fluid(isoprop, vol(900, UL), eppendorf2);
	vortex(eppendorf2);

	//13. Centrifuge at max speed for 10 min at RT.
	next_step();
	centrifuge_pellet(eppendorf2, speed(SPEED_MAX, RPM), RT, time(10, MINS));

	//14. Rinse the pellet with 1ml 70%EtOH.
	next_step();
	measure_fluid(etoh70, vol(1, ML), eppendorf2);
	vortex(eppendorf2);

	//15. Centrifuge at max speed for 5 min at RT.
	next_step();
	centrifuge_pellet(eppendorf2, speed(SPEED_MAX, RPM), RT, time(5, MINS));

	//16. Air dry the pellet.
	next_step();
	dry_pellet(eppendorf2, "in air");

	//17. Dissolve each in 50μL TE OR H2O. 
	next_step();
	first_option();
	measure_fluid(te, vol(50, UL), eppendorf2);
	next_option();
	measure_fluid(water, vol(50, UL), eppendorf2);
	end_option();
	dissolve(eppendorf2);

	end_protocol();
}
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