Small Scale Plasmid Isolation (Maxiprep) protocol - source code: Difference between revisions
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(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...) |
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<code> | <code> | ||
<pre> | <pre> | ||
#include " | #include "BioCoder.h" | ||
void main() | void main() | ||
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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( | 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); | ||
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// 6. Centrifuge for 5 minutes. | // 6. Centrifuge for 5 minutes. | ||
next_sub_step(); | next_sub_step(); | ||
centrifuge_phases_top(tube1, speed(SPEED_MAX, RPM), RT, time(5, MINS), tube2); | |||
//Phenol/Chloroform Cleanup: | //Phenol/Chloroform Cleanup: | ||
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// 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(tube2, phenol, 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. | ||
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next_sub_step(); | next_sub_step(); | ||
vortex(tube2); | vortex(tube2); | ||
centrifuge_phases_top(tube2, speed(SPEED_MAX, RPM), RT, time(5, MINS), tube3); | |||
//RNase (optional): | //RNase (optional): | ||
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// 3. Repeat the phenol/chloroform step. | // 3. Repeat the phenol/chloroform step. | ||
next_sub_step(); | next_sub_step(); | ||
measure_prop(tube3, phenol, 0.5); | |||
measure_prop(tube3, chloroform, 0.5); | |||
vortex(tube3); | vortex(tube3); | ||
centrifuge_phases_top(tube3, speed(SPEED_MAX, RPM), RT, time(5, MINS), tube4); | |||
//Alcohol Precipitation / Purification: | //Alcohol Precipitation / Purification: | ||
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// 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(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. |
Latest revision as of 22:59, 19 November 2009
#include "BioCoder.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", vol(2, UL));
Fluid isoprop = new_fluid("isopropanol", ICE_COLD);
Fluid etoh70 = new_fluid("70% ethanol");
Fluid water = new_fluid("sterile distilled water");
Container tube1 = new_container(CENTRI_TUBE_50ML);
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_top(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(tube2, phenol, 0.5);
measure_prop(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_top(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(tube3, phenol, 0.5);
measure_prop(tube3, chloroform, 0.5);
vortex(tube3);
centrifuge_phases_top(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(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();
}