Ethanol precipitation of small DNA fragments protocol - source code

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Revision as of 00:40, 20 November 2009 by Vaishnavi Ananth (Talk | contribs)
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#include "BioCoder.h"


void main()
{
	start_protocol("Ethanol Precipitation of small DNA fragments");

	Fluid ethanol = new_fluid("absolute ethanol", -20);
	Fluid dna = new_fluid("DNA sample",vol(20, UL));
	Fluid eth95 = new_fluid("95% ethanol", RT);
	Fluid water = new_fluid("water", vol(10, UL));

	Container eppendorf1 = new_container(EPPENDORF, dna);

	//1. Add 2 volumes ice cold absolute ethanol to sample.
	first_step();
	measure_prop(eppendorf1, ethanol, 2);
	comment("Generally the sample is in a 1.5 mL eppendorf tube. I recommend storing the absolute ethanol at -20°C.");

	//2. Incubate 1 hr at -80°C.
	next_step();
	incubate(eppendorf1, -80, time(1, HRS));
	comment("The long incubation time is critical for small fragments.");

	//3. Centrifuge for 30 minutes at 0°C at maximum speed (generally >10000 g at least).
	//4. Remove supernatant.
	next_step();
	centrifuge_pellet(eppendorf1, min_speed(10000, G), 0, time(30, MINS));

	//5. Wash with 750-1000 μL room-temperature 95% ethanol.
	next_step();
	measure_fluid(eth95, vol_range(750, 1000, UL), eppendorf1);
	comment("Another critical step for small fragments under 200 base pairs. Generally washing involves adding the ethanol and inverting several times.");

	//6. Centrifuge for 10 minutes at 4°C at maximum speed (generally >10000 g at least).
	next_step();
	centrifuge_pellet(eppendorf1, min_speed(10000, G), 4, time(10, MINS));

	//7. Let air dry on benchtop.
	next_step();
	dry_pellet(eppendorf1, "in air");
	comment("I generally let the pellet air dry completely such that it becomes white so that all residual ethanol is eliminated.");

	//8. Resuspend in an appropriate volume of H2O.
	next_step();
	measure_fluid(water, eppendorf1);
	comment("Use appropriate volume of water.");
	resuspend(eppendorf1);
	comment("Many protocols recommend resuspending in 10 mM Tris-HCl or TE. The advantage of TE is that EDTA chelates magnesium ions which makes it more difficult for residual DNases to degrade the DNA. I generally prefer H2O and don't seem to experience problems of this sort. If you plan to ultimately use electroporation to transform your DNA then resuspending in H2O has the advantage of keeping the salt content of your ligation reaction down.");

	end_protocol();
}
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