Small Scale Plasmid Isolation (Maxiprep) protocol - source code - Revision history

Vaishnavi Ananth at 05:59, 20 November 2009

2009-11-20T05:59:38Z

←Older revision		Revision as of 05:59, 20 November 2009
Line 13:		Line 13:
	Fluid phenol = new_fluid("phenol");		Fluid phenol = new_fluid("phenol");
	Fluid chloroform = new_fluid("chloroform");		Fluid chloroform = new_fluid("chloroform");
-	Fluid rnase = new_fluid("RNase to a final concentration of 3µg/ml");	+	Fluid rnase = new_fluid("RNase to a final concentration of 3µg/ml", vol(2, UL));
	Fluid isoprop = new_fluid("isopropanol", ICE_COLD);		Fluid isoprop = new_fluid("isopropanol", ICE_COLD);
	Fluid etoh70 = new_fluid("70% ethanol");		Fluid etoh70 = new_fluid("70% ethanol");
	Fluid water = new_fluid("sterile distilled water");		Fluid water = new_fluid("sterile distilled water");

-	Container tube1 = new_container(~~CENTRIFUGE_BOTTLE~~);	+	Container tube1 = new_container(CENTRI_TUBE_50ML);
	Container tube2 = new_container(CENTRIFUGE_TUBE_15ML);		Container tube2 = new_container(CENTRIFUGE_TUBE_15ML);
	Container tube3 = new_container(CENTRIFUGE_TUBE_15ML);		Container tube3 = new_container(CENTRIFUGE_TUBE_15ML);
Line 49:		Line 49:
	// 6. Centrifuge for 5 minutes.		// 6. Centrifuge for 5 minutes.
	next_sub_step();		next_sub_step();
-	~~centrifuge_phases~~(tube1, speed(SPEED_MAX, RPM), RT, time(5, MINS), tube2);	+	centrifuge_phases_top(tube1, speed(SPEED_MAX, RPM), RT, time(5, MINS), tube2);

	//Phenol/Chloroform Cleanup:		//Phenol/Chloroform Cleanup:
Line 56:		Line 56:
	// 2. Add 0.5 volumes of phenol and 0.5 volumes of chloroform to 1 volume of supernatant ~250:250->500µL (2.5:2.5->5mL).		// 2. Add 0.5 volumes of phenol and 0.5 volumes of chloroform to 1 volume of supernatant ~250:250->500µL (2.5:2.5->5mL).
	first_sub_step();		first_sub_step();
-	~~measure_prop_and_add~~(tube2, phenol, 0.5);	+	measure_prop(tube2, phenol, 0.5);
-	~~measure_prop_and_add~~(tube2, chloroform, 0.5);	+	measure_prop(tube2, chloroform, 0.5);

	// 3. Mix well by vortexing and centrifuge for 5 minutes.		// 3. Mix well by vortexing and centrifuge for 5 minutes.
Line 63:		Line 63:
	next_sub_step();		next_sub_step();
	vortex(tube2);		vortex(tube2);
-	~~centrifuge_phases~~(tube2, speed(SPEED_MAX, RPM), RT, time(5, MINS), tube3);	+	centrifuge_phases_top(tube2, speed(SPEED_MAX, RPM), RT, time(5, MINS), tube3);

	//RNase (optional):		//RNase (optional):
Line 78:		Line 78:
	// 3. Repeat the phenol/chloroform step.		// 3. Repeat the phenol/chloroform step.
	next_sub_step();		next_sub_step();
-	~~measure_prop_and_add~~(tube3, phenol, 0.5);	+	measure_prop(tube3, phenol, 0.5);
-	~~measure_prop_and_add~~(tube3, chloroform, 0.5);	+	measure_prop(tube3, chloroform, 0.5);
	vortex(tube3);		vortex(tube3);
-	~~centrifuge_phases~~(tube3, speed(SPEED_MAX, RPM), RT, time(5, MINS), tube4);	+	centrifuge_phases_top(tube3, speed(SPEED_MAX, RPM), RT, time(5, MINS), tube4);

	//Alcohol Precipitation / Purification:		//Alcohol Precipitation / Purification:
Line 87:		Line 87:
	// 1. Add an equal volume of ice cold isopropanol to the aqueous solution.		// 1. Add an equal volume of ice cold isopropanol to the aqueous solution.
	first_sub_step();		first_sub_step();
-	~~measure_prop_and_add~~(tube4, isoprop, 1);	+	measure_prop(tube4, isoprop, 1);

	// 2. Mix well by inversion and leave on ice for 10 minutes.		// 2. Mix well by inversion and leave on ice for 10 minutes.

Vaishnavi Ananth at 10:00, 19 November 2009

2009-11-19T10:00:21Z

←Older revision		Revision as of 10:00, 19 November 2009
Line 1:		Line 1:
	<code>		<code>
	<pre>		<pre>
-	#include "~~BioStream~~.h"	+	#include "BioCoder.h"

	void main()		void main()

Vaishnavi Ananth: New page:

      #include "BioStream.h"  void main() { 	start_protocol("Small-scale plasmid isolation - Maxiprep");  	Fluid culture = new_fluid("overnight culture"); 	Fluid sol1 = new_flui...


2009-10-23T07:54:30Z
New page: <code>     <pre> #include "BioStream.h"  void main() { 	start_protocol("Small-scale plasmid isolation - Maxiprep");  	Fluid culture = new_fluid("overnight culture"); 	Fluid sol1 = new_flui...
New page
<code>

    <pre>

#include "BioStream.h"



void main()

{

	start_protocol("Small-scale plasmid isolation - Maxiprep");



	Fluid culture = new_fluid("overnight culture");

	Fluid sol1 = new_fluid("Solution I", ICE_COLD);

	Fluid sol2 = new_fluid("Solution II");

	Fluid sol3 = new_fluid("Solution III");

	Fluid phenol = new_fluid("phenol");

	Fluid chloroform = new_fluid("chloroform");

	Fluid rnase = new_fluid("RNase to a final concentration of 3µg/ml");

	Fluid isoprop = new_fluid("isopropanol", ICE_COLD);

	Fluid etoh70 = new_fluid("70% ethanol");

	Fluid water = new_fluid("sterile distilled water");



	Container tube1 = new_container(CENTRIFUGE_BOTTLE);

	Container tube2 = new_container(CENTRIFUGE_TUBE_15ML);

	Container tube3 = new_container(CENTRIFUGE_TUBE_15ML);

	Container tube4 = new_container(CENTRIFUGE_TUBE_15ML);



	//Cell lysis:

	first_step("Cell Lysis");

	//   1. Take the overnight culture and spin down 1.5mL (50mL) of cells for 3 (5) minutes.

	first_sub_step();

	measure_fluid(culture, vol(50, ML), tube1);

	centrifuge_pellet(tube1, speed(SPEED_MAX, G), RT, time(5, MINS));



	//   3. Re-suspend the pellet in 100µL (1mL) of ice cold Solution I.

	next_sub_step();

	measure_fluid(sol1, vol(1, ML), tube1);

	resuspend(tube1);



	//   4. Add 200µL (2mL) of Solution II and gently invert the tube (roll the falcon) to mix. Leave on ice for 5 minutes.

	next_sub_step();

	measure_fluid(sol2, vol(2, ML), tube1);

	invert(tube1);

	store_for(tube1, ON_ICE, time(5, MINS));



	//   5. Add 150µL (1.5mL) of Solution III and shake vigorously. Leave on ice for 10 minutes.

	next_sub_step();

	measure_fluid(sol3, vol(1.5, ML), tube1);

	vortex(tube1);

	store_for(tube1, ON_ICE, time(10, MINS));



	//   6. Centrifuge for 5 minutes. 

	next_sub_step();

	centrifuge_phases(tube1, speed(SPEED_MAX, RPM), RT, time(5, MINS), tube2);



	//Phenol/Chloroform Cleanup:

	next_step("Phenol/Chloroform Cleanup");

	//   1. Transfer the supernatant by pipetting to a clean MCT (15mL falcon).

	//   2. Add 0.5 volumes of phenol and 0.5 volumes of chloroform to 1 volume of supernatant ~250:250->500µL (2.5:2.5->5mL).

	first_sub_step();

	measure_prop_and_add(tube2, phenol, 0.5);

	measure_prop_and_add(tube2, chloroform, 0.5);



	//   3. Mix well by vortexing and centrifuge for 5 minutes.

	//   4. Carefully take the top aqueous phase and transfer to a new tube. 

	next_sub_step();

	vortex(tube2);

	centrifuge_phases(tube2, speed(SPEED_MAX, RPM), RT, time(5, MINS), tube3);



	//RNase (optional):

	optional_step("RNase");

	//   1. Add RNase to a final concentration of 3µg/mL (5µL of 3mg/mL diluted stock / 5µL of 30mg/mL stock).

	first_sub_step();

	measure_fluid(rnase, tube3);



	//   2. Incubate in a 37°C water bath for 30-45 minutes.

	next_sub_step();

	incubate(tube3, 37, time_range(30, 45, MINS));

	comment("Use a water bath.");



	//   3. Repeat the phenol/chloroform step. 

	next_sub_step();

	measure_prop_and_add(tube3, phenol, 0.5);

	measure_prop_and_add(tube3, chloroform, 0.5);

	vortex(tube3);

	centrifuge_phases(tube3, speed(SPEED_MAX, RPM), RT, time(5, MINS), tube4);



	//Alcohol Precipitation / Purification:

	next_step("Alcohol Precipitation / Purification");

	//   1. Add an equal volume of ice cold isopropanol to the aqueous solution.

	first_sub_step();

	measure_prop_and_add(tube4, isoprop, 1);



	//   2. Mix well by inversion and leave on ice for 10 minutes.

	next_sub_step();

	invert(tube4);

	store_for(tube4, ON_ICE, time(10, MINS));



	//   3. Centrifuge at maximum speed for 15 minutes.

	//   4. Carefully pour off the isopropanol leaving the DNA pellet.

	next_sub_step();

	centrifuge_pellet(tube4, speed(SPEED_MAX, RPM), RT, time(15, MINS));





	//   5. Wash the pellet with 200µL (2mL) of 70% ethanol by pipetting.

	next_sub_step();

	measure_fluid(etoh70, vol(2, ML), tube4);

	pipet(tube4);



	//   6. Centrifuge for 5 minutes.

	next_sub_step();

	centrifuge_pellet(tube4, speed(SPEED_MAX, G), RT, time(5, MINS));



	//   7. Remove all the alcohol by pipetting and air dry the pellet for up to 30 minutes.

	next_sub_step();

	dry_pellet(tube4, "in air", max_time(30, MINS));



	//   8. Re-suspend the DNA pellet in 30-50µL (200-300µL) sdH2O.

	next_sub_step();

	measure_fluid(water, vol_range(200, 300, UL), tube4);

	resuspend(tube4);



	//   9. Store the samples at -20°C. 

	next_sub_step();

	store(tube4, -20);



	end_protocol();

}

    </pre>

</code>