Maxiprep of plasmid DNA from E.coli protocol - source code: Difference between revisions
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start_protocol("Maxiprep of Plasmid DNA from E.coli"); | start_protocol("Maxiprep of Plasmid DNA from E.coli"); | ||
Fluid medium = new_fluid("LB broth + selective marker"); | Fluid medium = new_fluid("LB broth + selective marker", vol(50, ML)); | ||
Fluid glycerol = new_fluid("50% sterile glycerol"); | Fluid glycerol = new_fluid("50% sterile glycerol"); | ||
Fluid teg = new_fluid("TEG", "25mM Tris-Cl, 10mM EDTA, 50mM dextrose"); | Fluid teg = new_fluid("TEG", "25mM Tris-Cl, 10mM EDTA, 50mM dextrose"); | ||
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Solid bac_col = new_solid("a single colony of E. coli"); | Solid bac_col = new_solid("a single colony of E. coli"); | ||
Container flask = new_container(FLASK); | Container flask = new_container(FLASK, medium); | ||
Container eppendorf1 = new_container(EPPENDORF); | Container eppendorf1 = new_container(EPPENDORF); | ||
Container eppendorf2 = new_container(EPPENDORF); | Container eppendorf2 = new_container(EPPENDORF); | ||
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// 1. Grow a single colony of E. coli overnight in 50mL LB broth + selective markers at 37°C. | // 1. Grow a single colony of E. coli overnight in 50mL LB broth + selective markers at 37°C. | ||
first_step(); | first_step(); | ||
inoculation(flask, bac_col, 37, time(12, HRS), 1); | inoculation(flask, bac_col, 37, time(12, HRS), 1); | ||
// 2. The next morning, put 850μL of the culture in each of two Eppendorf tubes, add 150μL sterile 50% glycerol, and store at -80°C. Pour as much culture as will fit into an Oak Ridge tube and centrifuge at 5800g/6000rpm, 4°C, for 10 minutes in a GSA rotor. Discard the supernatant, add the rest of the culture, and repeat. Resuspend in 1mL TEG. | // 2. The next morning, put 850μL of the culture in each of two Eppendorf tubes, add 150μL sterile 50% glycerol, and store at -80°C. Pour as much culture as will fit into an Oak Ridge tube and centrifuge at 5800g/6000rpm, 4°C, for 10 minutes in a GSA rotor. Discard the supernatant, add the rest of the culture, and repeat. Resuspend in 1mL TEG. | ||
next_step(); | next_step(); | ||
measure_fluid(flask, vol(850, UL), eppendorf1); | measure_fluid(flask, vol(850, UL), eppendorf1); | ||
measure_fluid(glycerol, vol(150, UL), eppendorf1); | |||
store(eppendorf1, -80); | store(eppendorf1, -80); | ||
measure_fluid(flask, vol(850, UL), eppendorf2); | measure_fluid(flask, vol(850, UL), eppendorf2); | ||
measure_fluid(glycerol, vol(150, UL), eppendorf2); | |||
store(eppendorf2, -80); | store(eppendorf2, -80); | ||
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centrifuge_pellet(oakridge, speed(5800, G), 4, time(10, MINS)); | centrifuge_pellet(oakridge, speed(5800, G), 4, time(10, MINS)); | ||
name_sample(flask, "rest of the culture"); | name_sample(flask, "rest of the culture"); | ||
measure_fluid(flask.contents, oakridge); | |||
centrifuge_pellet(oakridge, speed(5800, G), 4, time(10, MINS)); | centrifuge_pellet(oakridge, speed(5800, G), 4, time(10, MINS)); | ||
measure_fluid(teg, vol(1, ML), oakridge); | |||
resuspend(oakridge); | resuspend(oakridge); | ||
// 3. Add 111μL 20mg/mL lysozyme. Incubate on ice for 30 minutes. Meanwhile, mix: 250μL 10% SDS, 125μL 4M NaOH, 2.125mL autoclaved water per culture. | // 3. Add 111μL 20mg/mL lysozyme. Incubate on ice for 30 minutes. Meanwhile, mix: 250μL 10% SDS, 125μL 4M NaOH, 2.125mL autoclaved water per culture. | ||
next_step(); | next_step(); | ||
measure_fluid(lysozyme, vol(111, UL), oakridge); | |||
incubate(oakridge, ON_ICE, time(30, MINS)); | incubate(oakridge, ON_ICE, time(30, MINS)); | ||
parallel_step(); | parallel_step(); | ||
measure_fluid(sds, vol(250, UL), eppendorf3); | measure_fluid(sds, vol(250, UL), eppendorf3); | ||
measure_fluid(naoh, vol(125, UL), eppendorf3); | |||
measure_fluid(water, vol(2.125, ML), eppendorf3); | |||
vortex(eppendorf3); | vortex(eppendorf3); | ||
name_sample(eppendorf3, "SDS/NaOH mix"); | name_sample(eppendorf3, "SDS/NaOH mix"); | ||
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// 5. Add 1.5mL Solution 3 (3M K+, 5M acetate). Incubate on ice for 10 minutes. | // 5. Add 1.5mL Solution 3 (3M K+, 5M acetate). Incubate on ice for 10 minutes. | ||
next_step(); | next_step(); | ||
measure_fluid(sol3, vol(1.5, ML), oakridge); | |||
incubate(oakridge, ON_ICE, time(10, MINS)); | incubate(oakridge, ON_ICE, time(10, MINS)); | ||
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next_step(); | next_step(); | ||
vortex(oakridge); | vortex(oakridge); | ||
centrifuge_phases_top(oakridge, speed(17200, G), 4, time(15, MINS), oakridge1); | |||
// 7. Pour the supernatant into another Oak Ridge tube and discard the pellet. Add 2.7mL isopropanol. Centrifuge at 17,200g/12,000rpm (room temperature) for 10 minutes. Discard the supernatant. | // 7. Pour the supernatant into another Oak Ridge tube and discard the pellet. Add 2.7mL isopropanol. Centrifuge at 17,200g/12,000rpm (room temperature) for 10 minutes. Discard the supernatant. | ||
next_step(); | next_step(); | ||
measure_fluid(isoprop, vol(2.7, ML), oakridge1); | |||
centrifuge_pellet(oakridge1, speed(17200, G), RT, time(10, MINS)); | centrifuge_pellet(oakridge1, speed(17200, G), RT, time(10, MINS)); | ||
// 8. Wash pellet with 1mL 70% ethanol. Air dry for 2-5 minutes on bench. Resuspend in 500μL TE buffer. Add 500μL 5M LiCl. Incubate on ice for 5 minutes. | // 8. Wash pellet with 1mL 70% ethanol. Air dry for 2-5 minutes on bench. Resuspend in 500μL TE buffer. Add 500μL 5M LiCl. Incubate on ice for 5 minutes. | ||
next_step(); | next_step(); | ||
measure_fluid(eth70, vol(1, ML), oakridge1); | |||
vortex(oakridge1); | vortex(oakridge1); | ||
centrifuge_pellet(oakridge1, speed(17200, G), RT, time(10, MINS)); | centrifuge_pellet(oakridge1, speed(17200, G), RT, time(10, MINS)); | ||
dry_pellet(oakridge1, "in air", time_range(2, 5, MINS)); | dry_pellet(oakridge1, "in air", time_range(2, 5, MINS)); | ||
measure_fluid(te, vol(500, UL), oakridge1); | |||
resuspend(oakridge1); | resuspend(oakridge1); | ||
measure_fluid(licl, vol(500, UL), oakridge1); | |||
incubate(oakridge1, ON_ICE, time(5, MINS)); | incubate(oakridge1, ON_ICE, time(5, MINS)); | ||
// 9. Centrifuge at 17,200g/12,000rpm for 10 minutes. | // 9. Centrifuge at 17,200g/12,000rpm for 10 minutes. | ||
centrifuge_phases_top(oakridge1, speed(17200, G), RT, time(10, MINS), eppendorf4); | |||
//10. Pour supernatant into an Eppendorf. Add 1mL isopropanol. Incubate on the bench for 10 minutes. | //10. Pour supernatant into an Eppendorf. Add 1mL isopropanol. Incubate on the bench for 10 minutes. | ||
next_step(); | next_step(); | ||
measure_fluid(isoprop, vol(1, ML), eppendorf4); | |||
incubate(eppendorf4, RT, time(10, MINS)); | incubate(eppendorf4, RT, time(10, MINS)); | ||
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//12. Discard the supernatant. Wash the pellets with 100μL 70% ethanol. Resuspend in 375μL TE buffer. Add 7.5μL 1mg/mL RNaseA. Incubate at 37°C for 30 minutes. | //12. Discard the supernatant. Wash the pellets with 100μL 70% ethanol. Resuspend in 375μL TE buffer. Add 7.5μL 1mg/mL RNaseA. Incubate at 37°C for 30 minutes. | ||
next_step(); | next_step(); | ||
measure_fluid(eth70, vol(100, UL), eppendorf4); | |||
vortex(eppendorf4); | vortex(eppendorf4); | ||
centrifuge_pellet(eppendorf4, speed(17200, G), RT, time(10, MINS)); | centrifuge_pellet(eppendorf4, speed(17200, G), RT, time(10, MINS)); | ||
measure_fluid(te, vol(375, UL), eppendorf4); | |||
resuspend(eppendorf4); | resuspend(eppendorf4); | ||
measure_fluid(rnase, vol(7.5, UL), eppendorf4); | |||
incubate(eppendorf4, 37, time(30, MINS)); | incubate(eppendorf4, 37, time(30, MINS)); | ||
//13. Add 700μL phenol:chloroform:isoamyl alcohol. Vortex until thoroughly mixed. Centrifuge at top speed of microfuge for 2 minutes. Pipette aqueous phase (the top one) into new Eppendorf. Repeat until the interface between the phases is clear after centrifugation. Then repeat the procedure twice with chloroform:isoamyl alcohol to remove any phenol. | //13. Add 700μL phenol:chloroform:isoamyl alcohol. Vortex until thoroughly mixed. Centrifuge at top speed of microfuge for 2 minutes. Pipette aqueous phase (the top one) into new Eppendorf. Repeat until the interface between the phases is clear after centrifugation. Then repeat the procedure twice with chloroform:isoamyl alcohol to remove any phenol. | ||
next_step(); | next_step(); | ||
measure_fluid(phe_chloro_iaa, vol(700, UL), eppendorf4); | |||
vortex(eppendorf4); | vortex(eppendorf4); | ||
comment("The solution should be thoroughly mixed."); | comment("The solution should be thoroughly mixed."); | ||
centrifuge_phases_top(eppendorf4, speed(SPEED_MAX, G), RT, time(2, MINS), eppendorf5); | |||
next_step(); | next_step(); | ||
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next_step(); | next_step(); | ||
measure_fluid(chloro_iaa, vol(700, UL), eppendorf5); | |||
vortex(eppendorf5); | vortex(eppendorf5); | ||
comment("The solution should be thoroughly mixed."); | comment("The solution should be thoroughly mixed."); | ||
centrifuge_phases_top(eppendorf5, speed(SPEED_MAX, G), RT, time(2, MINS), eppendorf6); | |||
next_step(); | next_step(); | ||
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//14. Add 750μL straight ethanol and 125μL 3M sodium acetate. Put at -80°C for 30 minutes or -20°C overnight. | //14. Add 750μL straight ethanol and 125μL 3M sodium acetate. Put at -80°C for 30 minutes or -20°C overnight. | ||
next_step(); | next_step(); | ||
measure_fluid(eth100, vol(750, UL), eppendorf6); | |||
measure_fluid(naac, vol(125, UL), eppendorf6); | |||
first_option(); | |||
store_for(eppendorf6, -80, time(30, MINS)); | store_for(eppendorf6, -80, time(30, MINS)); | ||
next_option(); | next_option(); | ||
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next_step(); | next_step(); | ||
centrifuge_pellet(eppendorf6, speed(13600, G), 4, time(15, MINS)); | centrifuge_pellet(eppendorf6, speed(13600, G), 4, time(15, MINS)); | ||
measure_fluid(eth70, approx_vol(100, UL), eppendorf6); | |||
vortex(eppendorf6); | vortex(eppendorf6); | ||
centrifuge_pellet(eppendorf6, speed(13600, G), 4, time(5, MINS)); | centrifuge_pellet(eppendorf6, speed(13600, G), 4, time(5, MINS)); | ||
measure_fluid(te, vol_range(100, 200, UL), eppendorf6); | |||
resuspend(eppendorf6); | resuspend(eppendorf6); | ||
Revision as of 22:35, 19 November 2009
#include "BioCoder.h"
void main()
{
start_protocol("Maxiprep of Plasmid DNA from E.coli");
Fluid medium = new_fluid("LB broth + selective marker", vol(50, ML));
Fluid glycerol = new_fluid("50% sterile glycerol");
Fluid teg = new_fluid("TEG", "25mM Tris-Cl, 10mM EDTA, 50mM dextrose");
Fluid lysozyme = new_fluid("20 mg/ml lysozyme");
Fluid sds = new_fluid("10% SDS");
Fluid naoh = new_fluid("4M NaOH");
Fluid water = new_fluid("autoclaved water");
Fluid sol3 = new_fluid("Solution 3", "3M potassium-acetate, 2M acetic acid -- glacial is 17M");
Fluid isoprop = new_fluid("isopropanol");
Fluid eth70 = new_fluid("70% ethanol");
Fluid te = new_fluid("TE buffer");
Fluid licl = new_fluid("5M LiCl");
Fluid rnase = new_fluid("1 mg/ml RNaseA");
Fluid phe_chloro_iaa = new_fluid("phenol: chloroform: isoamyl alcohol", "25:24:1");
Fluid chloro_iaa = new_fluid("chloroform: isoamyl alcohol", "24:1");
Fluid eth100 = new_fluid("straight ethanol");
Fluid naac = new_fluid("3M sodium acetate");
Solid bac_col = new_solid("a single colony of E. coli");
Container flask = new_container(FLASK, medium);
Container eppendorf1 = new_container(EPPENDORF);
Container eppendorf2 = new_container(EPPENDORF);
Container eppendorf3 = new_container(EPPENDORF);
Container eppendorf4 = new_container(EPPENDORF);
Container eppendorf5 = new_container(EPPENDORF);
Container eppendorf6 = new_container(EPPENDORF);
Container oakridge = new_container(OAKRIDGE);
Container oakridge1 = new_container(OAKRIDGE);
// 1. Grow a single colony of E. coli overnight in 50mL LB broth + selective markers at 37°C.
first_step();
inoculation(flask, bac_col, 37, time(12, HRS), 1);
// 2. The next morning, put 850μL of the culture in each of two Eppendorf tubes, add 150μL sterile 50% glycerol, and store at -80°C. Pour as much culture as will fit into an Oak Ridge tube and centrifuge at 5800g/6000rpm, 4°C, for 10 minutes in a GSA rotor. Discard the supernatant, add the rest of the culture, and repeat. Resuspend in 1mL TEG.
next_step();
measure_fluid(flask, vol(850, UL), eppendorf1);
measure_fluid(glycerol, vol(150, UL), eppendorf1);
store(eppendorf1, -80);
measure_fluid(flask, vol(850, UL), eppendorf2);
measure_fluid(glycerol, vol(150, UL), eppendorf2);
store(eppendorf2, -80);
next_step();
name_sample(flask, "as much culture as will fit");
measure_fluid(flask.contents, oakridge);
centrifuge_pellet(oakridge, speed(5800, G), 4, time(10, MINS));
name_sample(flask, "rest of the culture");
measure_fluid(flask.contents, oakridge);
centrifuge_pellet(oakridge, speed(5800, G), 4, time(10, MINS));
measure_fluid(teg, vol(1, ML), oakridge);
resuspend(oakridge);
// 3. Add 111μL 20mg/mL lysozyme. Incubate on ice for 30 minutes. Meanwhile, mix: 250μL 10% SDS, 125μL 4M NaOH, 2.125mL autoclaved water per culture.
next_step();
measure_fluid(lysozyme, vol(111, UL), oakridge);
incubate(oakridge, ON_ICE, time(30, MINS));
parallel_step();
measure_fluid(sds, vol(250, UL), eppendorf3);
measure_fluid(naoh, vol(125, UL), eppendorf3);
measure_fluid(water, vol(2.125, ML), eppendorf3);
vortex(eppendorf3);
name_sample(eppendorf3, "SDS/NaOH mix");
// 4. Add 2mL SDS/NaOH mix to each tube. Incubate on ice for 10 minutes.
next_step();
measure_fluid(eppendorf3, vol(2, ML), oakridge);
incubate(oakridge, ON_ICE, time(10, MINS));
// 5. Add 1.5mL Solution 3 (3M K+, 5M acetate). Incubate on ice for 10 minutes.
next_step();
measure_fluid(sol3, vol(1.5, ML), oakridge);
incubate(oakridge, ON_ICE, time(10, MINS));
// 6. Shake vigorously. Centrifuge in SS34 rotor at 17,200g/12,000rpm, 4°C, for 15 minutes.
next_step();
vortex(oakridge);
centrifuge_phases_top(oakridge, speed(17200, G), 4, time(15, MINS), oakridge1);
// 7. Pour the supernatant into another Oak Ridge tube and discard the pellet. Add 2.7mL isopropanol. Centrifuge at 17,200g/12,000rpm (room temperature) for 10 minutes. Discard the supernatant.
next_step();
measure_fluid(isoprop, vol(2.7, ML), oakridge1);
centrifuge_pellet(oakridge1, speed(17200, G), RT, time(10, MINS));
// 8. Wash pellet with 1mL 70% ethanol. Air dry for 2-5 minutes on bench. Resuspend in 500μL TE buffer. Add 500μL 5M LiCl. Incubate on ice for 5 minutes.
next_step();
measure_fluid(eth70, vol(1, ML), oakridge1);
vortex(oakridge1);
centrifuge_pellet(oakridge1, speed(17200, G), RT, time(10, MINS));
dry_pellet(oakridge1, "in air", time_range(2, 5, MINS));
measure_fluid(te, vol(500, UL), oakridge1);
resuspend(oakridge1);
measure_fluid(licl, vol(500, UL), oakridge1);
incubate(oakridge1, ON_ICE, time(5, MINS));
// 9. Centrifuge at 17,200g/12,000rpm for 10 minutes.
centrifuge_phases_top(oakridge1, speed(17200, G), RT, time(10, MINS), eppendorf4);
//10. Pour supernatant into an Eppendorf. Add 1mL isopropanol. Incubate on the bench for 10 minutes.
next_step();
measure_fluid(isoprop, vol(1, ML), eppendorf4);
incubate(eppendorf4, RT, time(10, MINS));
//11. Centrifuge at 17,200g/12,000rpm for 10 minutes.
next_step();
centrifuge_pellet(eppendorf4, speed(17200, G), RT, time(10, MINS));
//12. Discard the supernatant. Wash the pellets with 100μL 70% ethanol. Resuspend in 375μL TE buffer. Add 7.5μL 1mg/mL RNaseA. Incubate at 37°C for 30 minutes.
next_step();
measure_fluid(eth70, vol(100, UL), eppendorf4);
vortex(eppendorf4);
centrifuge_pellet(eppendorf4, speed(17200, G), RT, time(10, MINS));
measure_fluid(te, vol(375, UL), eppendorf4);
resuspend(eppendorf4);
measure_fluid(rnase, vol(7.5, UL), eppendorf4);
incubate(eppendorf4, 37, time(30, MINS));
//13. Add 700μL phenol:chloroform:isoamyl alcohol. Vortex until thoroughly mixed. Centrifuge at top speed of microfuge for 2 minutes. Pipette aqueous phase (the top one) into new Eppendorf. Repeat until the interface between the phases is clear after centrifugation. Then repeat the procedure twice with chloroform:isoamyl alcohol to remove any phenol.
next_step();
measure_fluid(phe_chloro_iaa, vol(700, UL), eppendorf4);
vortex(eppendorf4);
comment("The solution should be thoroughly mixed.");
centrifuge_phases_top(eppendorf4, speed(SPEED_MAX, G), RT, time(2, MINS), eppendorf5);
next_step();
repeat(14, "the interface between the phases is clear after centrifugation");
next_step();
measure_fluid(chloro_iaa, vol(700, UL), eppendorf5);
vortex(eppendorf5);
comment("The solution should be thoroughly mixed.");
centrifuge_phases_top(eppendorf5, speed(SPEED_MAX, G), RT, time(2, MINS), eppendorf6);
next_step();
repeat(16);
comment("This removes phenol.");
//14. Add 750μL straight ethanol and 125μL 3M sodium acetate. Put at -80°C for 30 minutes or -20°C overnight.
next_step();
measure_fluid(eth100, vol(750, UL), eppendorf6);
measure_fluid(naac, vol(125, UL), eppendorf6);
first_option();
store_for(eppendorf6, -80, time(30, MINS));
next_option();
store_for(eppendorf6, -20, time(12, HRS));
end_option();
//15. Centrifuge at 13,600g/12,000rpm, 4°C, for 15 minutes. Discard the supernatant. Wash pellet with ~100μL 70% ethanol. Resuspend in 100-200μL TE buffer.
next_step();
centrifuge_pellet(eppendorf6, speed(13600, G), 4, time(15, MINS));
measure_fluid(eth70, approx_vol(100, UL), eppendorf6);
vortex(eppendorf6);
centrifuge_pellet(eppendorf6, speed(13600, G), 4, time(5, MINS));
measure_fluid(te, vol_range(100, 200, UL), eppendorf6);
resuspend(eppendorf6);
end_protocol();
}