Ethanol precipitation of small DNA fragments protocol - source code

#include "BioStream.h"


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

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

	Container eppendorf1 = new_container(EPPENDORF);

	//1. Add 2 volumes ice cold absolute ethanol to sample.
	first_step();
	measure_fluid(dna, eppendorf1);
	measure_prop_and_add(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_and_add(eppendorf1, eth95, vol_range(750, 1000, UL));
	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_and_add(eppendorf1, water);
	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 H<sub>2</sub>O and don't seem to experience problems of this sort. If you plan to ultimately use electroporation to transform your DNA then resuspending in H<sub>2</sub>O has the advantage of keeping the salt content of your ligation reaction down.");

	end_protocol();
}