Filamentous fungi genomic DNA isolation protocol - source code

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#include "BioCoder.h"

void main()
{

	start_protocol("Filamentous fungi genomic DNA isolation");

	Fluid lysis_buffer = new_fluid("lysis buffer", "50 mM Tris-HCL, 50 mM EDTA, 3% SDS, 1% 2-mercaptoethanol (add just before use)");
	Fluid mercaptor = new_fluid("B-mercaptor");
	Fluid sevag = new_fluid("SEVAG", "chloroform:isoamyl alcohol, 24:1");
	Fluid naoc = new_fluid("3M NaOAC", "pH 8.0");
	Fluid isoprop = new_fluid("isopropanol");
	Fluid eb = new_fluid("EB");
	Fluid rnase = new_fluid("100 mg/ml RNAse");
	Fluid amm_acetate = new_fluid("7.5 M ammonium acetate");
	Fluid ethanol100 = new_fluid("100% EtOH");
	Fluid ethanol70 = new_fluid("70% EtOH");
	Fluid te = new_fluid("TE buffer", "pH 8.0");

	Solid fungi = new_solid("group lyophilized mycelium(wet)", "ground in liquid nitrogen");

	Container sterile_microfuge_tube1 = new_container(STERILE_MICROFUGE_TUBE);
	Container eppendorf1 = new_container(EPPENDORF);
	Container eppendorf2 = new_container(EPPENDORF);
	Container eppendorf3 = new_container(EPPENDORF);

	// 1.  Fill a 1.5 mL eppendorf microcentrifuge tube 2/3 to the joint with group lyophilized mycelium (60-100 mg dry, or 0.5-1.0 g wet, ground in in liquid nitrogen)
	//        * Alternatively you can grind 1 g of dried (vacuum filter mycelium first) in a mortar and pestle treating with liquid nitrogen 5-6 times. our the frozen powder into the eppendorf tube.
	//        * Note: you only want to process about 1g in each eppendorf tube, if there is more than this, split to two separate tubes. 
	first_step();
	measure_solid(fungi, 1, GR, sterile_microfuge_tube1);
	comment("If using dry fungi, use 60- 100 mg. Alternatively you can grind 1 g of dried (vacuum filter mycelium first) in a mortar and pestle treating with liquid nitrogen 5-6 times. Pour the frozen powder into the eppendorf tube.");
	comment("Note: you only want to process about 1g in each eppendorf tube, if there is more than this, split to two separate tubes.");

	// 2. Add 660-750μL + 10 μL Β-mercaptor.
	//        * Vortex to insure good mixing of solution, incubate in 65 °C water bath for 1hr. 
	next_step();
	measure_fluid(lysis_buffer, vol_range(660, 750, UL), sterile_microfuge_tube1);
	measure_fluid(mercaptor, vol(10, UL), sterile_microfuge_tube1);
	vortex(sterile_microfuge_tube1);
	incubate(sterile_microfuge_tube1, 65, time(1, HRS));
	comment("Use a water bath for incubation.");

	// 3. Spin down to remove cell debris. 5min at 3400 RPM. Transfer aqueous phase (top) to 1.mL eppendorf. Do not take any of the cellular debris from the interface. Don't be greedy this prep extracts a lot of DNA.

	next_step();
	centrifuge_phases_top(sterile_microfuge_tube1, speed(3400, RPM), RT, time(5, MINS), eppendorf1);
	comment("Do not take any of the cellular debris from the interface. Don't be greedy this prep extracts a lot of DNA.");

	// 4. Add 700 μL of SEVAG; (adjust volume if needed to meet a 1:1 ratio of SEVAGE and aqueous phase) and vortex. Microcentrofuge at 12,000 g for 10 min.
	//        * Note: some people top off with EB buffer so that total volume is so that volume is equal among the samples for spinning. Be careful as sometimes tops are loosened by chloroform. 
	next_step();
	measure_fluid(sevag, vol(700, UL), eppendorf1);
	comment("Adjust volume if needed to meet a 1:1 ratio of SEVAG and aqueous phase.");
	vortex(eppendorf1);
	comment("Note: some people top off with EB buffer so that total volume is so that volume is equal among the samples for spinning. Be careful as sometimes tops are loosened by chloroform.");
	centrifuge_phases_top(eppendorf1, speed(12000, G), RT, time(10, MINS), vol_range(550, 600, UL), eppendorf2);

	// 5. Transfer aqueous phase (top) to new tube.
	//        * Note: these last two steps (SEVAG, spin, transfer) can be repeated to insure cleaner DNA prep depending on your needs and pipette techniques. 
	next_step();
	comment("Note: these last two steps (SEVAG, spin, transfer) can be repeated to insure cleaner DNA prep depending on your needs and pipette techniques.");

	// 6. Remove aqueous phase (top) to a new tube (approx. 550-600 μL). Add 20 μL of 3 M NaOAc. Top off with isopropanol. Invert gently. You should see DNA "ropes" precipitating.
	next_step();
	measure_fluid(naoc, vol(20, UL), eppendorf2);
	measure_fluid(isoprop, eppendorf2);
	comment("Top off with isopropanol.");
	invert(eppendorf2);
	comment("You should see DNA \"ropes\" precipitating.");

	// 7. Microcentrifuge for 2 min. Pour off supernatent (top layer). Invert tube for 1 min to dry.
	next_step();
	centrifuge_pellet(eppendorf2, speed(SPEED_MAX, RPM), RT, time(2, MINS));
	invert_dry(eppendorf2, RT, time(1, MINS));

	// 8. Add 300 μL EB (or ddH20) and 1 μL of 100 mg/mL RNAse and place in 65°C for 10-15 min. Finger vortex (you don't want to shear this nice long DNA now do you?).
	next_step();
	measure_fluid(eb, vol(300, UL), eppendorf2);
	measure_fluid(rnase, vol(1, UL), eppendorf2);
	store_for(eppendorf2, 65, time_range(10, 15, MINS));
	vortex(eppendorf2);
	comment("Finger vortex the tube to prevent shearing of DNA.");

	// 9. (Optional) further cleanup with the PEG DNA protocol OR
	optional_step();
	to_do("Further cleanup with the PEG DNA protocol.");

	//10. 250 μL 7.5M Ammonium acetate. Spin at max speed 5min to pellet protein debris. Take supernatent out, add 750 isopropanol.
	next_step();
	measure_fluid(amm_acetate, vol(250, UL), eppendorf2);
	centrifuge_phases_top(eppendorf2, speed(SPEED_MAX, RPM), RT, time(5, MINS), eppendorf3);
	comment("This pellets the protein debris.");
	measure_fluid(isoprop, vol(750, UL), eppendorf3);

	//11. Microcentrifuge 30s to 2 min to pellet the DNA. Pour off the supernatent and rinse the pellet with 100% EtOH. Wash again with 70% EtOH.
	next_step();
	centrifuge_pellet(eppendorf3, speed(SPEED_MAX, RPM), RT, time_range(30, 120, SECS));
	measure_fluid(ethanol100, vol(1, ML), eppendorf3);
	vortex(eppendorf3);
	centrifuge_pellet(eppendorf3, speed(SPEED_MAX, RPM), RT, time_range(30, 120, SECS));
	measure_fluid(ethanol70, vol(1, ML), eppendorf3);
	vortex(eppendorf3);
	centrifuge_pellet(eppendorf3, speed(SPEED_MAX, RPM), RT, time_range(30, 120, SECS));

	//12. Dry tubes in vacuum oven at 50°C for 15 min at most or dry in speed vac.
	next_step();
	first_option();
	dry_pellet(eppendorf3, "in vacuum oven at 50°C", max_time(15, MINS));
	next_option();
	dry_pellet(eppendorf3, "in speed vac");
	end_option();

	//13. Resuspend in pH 8 TE buffer. 
	next_step();
	measure_fluid(te, eppendorf3);
	resuspend(eppendorf3);

	end_protocol();
}
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