Erman's Lab:DNA Miniprep with Alkaline Lysis protocol - source code: Difference between revisions
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start_protocol("Erman's lab - DNA miniprep with alkaline lysis"); | start_protocol("Erman's lab - DNA miniprep with alkaline lysis"); | ||
Fluid lb = new_fluid(" | Fluid lb = new_fluid("LB (+ antibiotics)", vol(2, ML)); | ||
Fluid sln1 = new_fluid("Alkaline Lysis SLN1", "20mM Tris(pH 8), 50mM Glucose, 10mM EDTA"); | Fluid sln1 = new_fluid("Alkaline Lysis SLN1", "20mM Tris(pH 8), 50mM Glucose, 10mM EDTA"); | ||
Fluid al2 = new_fluid("freshly prepared AL2", "0.2N NaOH, 1% SDS"); | Fluid al2 = new_fluid("freshly prepared AL2", "0.2N NaOH, 1% SDS"); | ||
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Solid colony = new_solid("single colony"); | Solid colony = new_solid("single colony"); | ||
Container flask = new_container(FLASK); | Container flask = new_container(FLASK,lb); | ||
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. o/n grow single colony in 2ml of LB (+ antibiotics) | //1. o/n grow single colony in 2ml of LB (+ antibiotics) | ||
first_step(); | first_step(); | ||
inoculation(flask, colony, 37, time(12, HRS), 1); | inoculation(flask, colony, 37, time(12, HRS), 1); | ||
//2. 1.5 ml into eppendorf. | // 2. 1.5 ml into eppendorf. | ||
next_step(); | next_step(); | ||
measure_fluid(flask, vol(2, ML), eppendorf1); | measure_fluid(flask, vol(2, ML), eppendorf1); | ||
//3. Pellet cells at max speed in cold room for 1.5 min. | // 3. Pellet cells at max speed in cold room for 1.5 min. | ||
next_step(); | next_step(); | ||
centrifuge_pellet(eppendorf1, speed(SPEED_MAX, G), 4, time(1.5, MINS)); | centrifuge_pellet(eppendorf1, speed(SPEED_MAX, G), 4, time(1.5, MINS)); | ||
//4. Discard supernatant , leave pellet as dry as possible. | // 4. Discard supernatant , leave pellet as dry as possible. | ||
next_step(); | next_step(); | ||
comment("Leave pellet as dry as possible."); | comment("Leave pellet as dry as possible."); | ||
//5. Resuspend pellet in 100 μL Alkaline Lysis SLN1,vortex. | // 5. Resuspend pellet in 100 μL Alkaline Lysis SLN1,vortex. | ||
next_step(); | next_step(); | ||
measure_fluid(sln1, vol(100, UL), eppendorf1); | measure_fluid(sln1, vol(100, UL), eppendorf1); | ||
resuspend(eppendorf1); | resuspend(eppendorf1); | ||
//6. Add 200μL freshly prepared AL2, mix by inverting 5-6 times(DO NOT vortex!) | // 6. Add 200μL freshly prepared AL2, mix by inverting 5-6 times(DO NOT vortex!) | ||
next_step(); | next_step(); | ||
measure_fluid(al2, vol(200, UL), eppendorf1); | measure_fluid(al2, vol(200, UL), eppendorf1); | ||
Line 48: | Line 47: | ||
comment("Do not vortex!"); | comment("Do not vortex!"); | ||
//7. Put on ice. | // 7. Put on ice. | ||
next_step(); | next_step(); | ||
store(eppendorf1, ON_ICE); | store(eppendorf1, ON_ICE); | ||
//8. Add 300μL AL3,invert tubes to mix. | // 8. Add 300μL AL3,invert tubes to mix. | ||
next_step(); | next_step(); | ||
measure_fluid(al3, vol(300, UL), eppendorf1); | measure_fluid(al3, vol(300, UL), eppendorf1); | ||
invert(eppendorf1); | invert(eppendorf1); | ||
//9. Incubate on ice for 5 min. | // 9. Incubate on ice for 5 min. | ||
next_step(); | next_step(); | ||
incubate(eppendorf1, ON_ICE, time(5, MINS)); | incubate(eppendorf1, ON_ICE, time(5, MINS)); | ||
//10. Centrifuge at max speed for 5 min in cold room. | // 10. Centrifuge at max speed for 5 min in cold room. | ||
//11. Take the supernatant into a new tube. | // 11. Take the supernatant into a new tube. | ||
next_step(); | next_step(); | ||
centrifuge_phases_top(eppendorf1, speed(SPEED_MAX, RPM), 4, time(5, MINS), eppendorf2); | |||
//12. Add 900 isopropanol at RT. Vortex | // 12. Add 900 isopropanol at RT. Vortex | ||
next_step(); | next_step(); | ||
measure_fluid(isoprop, vol(900, UL), eppendorf2); | measure_fluid(isoprop, vol(900, UL), eppendorf2); | ||
vortex(eppendorf2); | vortex(eppendorf2); | ||
//13. Centrifuge at max speed for 10 min at RT. | // 13. Centrifuge at max speed for 10 min at RT. | ||
next_step(); | next_step(); | ||
centrifuge_pellet(eppendorf2, speed(SPEED_MAX, RPM), RT, time(10, MINS)); | centrifuge_pellet(eppendorf2, speed(SPEED_MAX, RPM), RT, time(10, MINS)); | ||
//14. Rinse the pellet with 1ml 70%EtOH. | // 14. Rinse the pellet with 1ml 70%EtOH. | ||
next_step(); | next_step(); | ||
measure_fluid(etoh70, vol(1, ML), eppendorf2); | measure_fluid(etoh70, vol(1, ML), eppendorf2); | ||
vortex(eppendorf2); | vortex(eppendorf2); | ||
//15. Centrifuge at max speed for 5 min at RT. | // 15. Centrifuge at max speed for 5 min at RT. | ||
next_step(); | next_step(); | ||
centrifuge_pellet(eppendorf2, speed(SPEED_MAX, RPM), RT, time(5, MINS)); | centrifuge_pellet(eppendorf2, speed(SPEED_MAX, RPM), RT, time(5, MINS)); | ||
//16. Air dry the pellet. | // 16. Air dry the pellet. | ||
next_step(); | next_step(); | ||
dry_pellet(eppendorf2, "in air"); | dry_pellet(eppendorf2, "in air"); | ||
//17. Dissolve each in 50μL TE OR H2O. | // 17. Dissolve each in 50μL TE OR H2O. | ||
next_step(); | next_step(); | ||
first_option(); | first_option(); |
Latest revision as of 22:49, 19 November 2009
#include "BioCoder.h"
void main()
{
start_protocol("Erman's lab - DNA miniprep with alkaline lysis");
Fluid lb = new_fluid("LB (+ antibiotics)", vol(2, ML));
Fluid sln1 = new_fluid("Alkaline Lysis SLN1", "20mM Tris(pH 8), 50mM Glucose, 10mM EDTA");
Fluid al2 = new_fluid("freshly prepared AL2", "0.2N NaOH, 1% SDS");
Fluid al3 = new_fluid("AL3", "3M KAc, glacial acetic acid");
Fluid isoprop = new_fluid("isopropanol", RT);
Fluid etoh70 = new_fluid("70% EtOH");
Fluid te = new_fluid("TE");
Fluid water = new_fluid("water");
Solid colony = new_solid("single colony");
Container flask = new_container(FLASK,lb);
Container eppendorf1 = new_container(EPPENDORF);
Container eppendorf2 = new_container(EPPENDORF);
//1. o/n grow single colony in 2ml of LB (+ antibiotics)
first_step();
inoculation(flask, colony, 37, time(12, HRS), 1);
// 2. 1.5 ml into eppendorf.
next_step();
measure_fluid(flask, vol(2, ML), eppendorf1);
// 3. Pellet cells at max speed in cold room for 1.5 min.
next_step();
centrifuge_pellet(eppendorf1, speed(SPEED_MAX, G), 4, time(1.5, MINS));
// 4. Discard supernatant , leave pellet as dry as possible.
next_step();
comment("Leave pellet as dry as possible.");
// 5. Resuspend pellet in 100 μL Alkaline Lysis SLN1,vortex.
next_step();
measure_fluid(sln1, vol(100, UL), eppendorf1);
resuspend(eppendorf1);
// 6. Add 200μL freshly prepared AL2, mix by inverting 5-6 times(DO NOT vortex!)
next_step();
measure_fluid(al2, vol(200, UL), eppendorf1);
invert(eppendorf1, 5, 6);
comment("Do not vortex!");
// 7. Put on ice.
next_step();
store(eppendorf1, ON_ICE);
// 8. Add 300μL AL3,invert tubes to mix.
next_step();
measure_fluid(al3, vol(300, UL), eppendorf1);
invert(eppendorf1);
// 9. Incubate on ice for 5 min.
next_step();
incubate(eppendorf1, ON_ICE, time(5, MINS));
// 10. Centrifuge at max speed for 5 min in cold room.
// 11. Take the supernatant into a new tube.
next_step();
centrifuge_phases_top(eppendorf1, speed(SPEED_MAX, RPM), 4, time(5, MINS), eppendorf2);
// 12. Add 900 isopropanol at RT. Vortex
next_step();
measure_fluid(isoprop, vol(900, UL), eppendorf2);
vortex(eppendorf2);
// 13. Centrifuge at max speed for 10 min at RT.
next_step();
centrifuge_pellet(eppendorf2, speed(SPEED_MAX, RPM), RT, time(10, MINS));
// 14. Rinse the pellet with 1ml 70%EtOH.
next_step();
measure_fluid(etoh70, vol(1, ML), eppendorf2);
vortex(eppendorf2);
// 15. Centrifuge at max speed for 5 min at RT.
next_step();
centrifuge_pellet(eppendorf2, speed(SPEED_MAX, RPM), RT, time(5, MINS));
// 16. Air dry the pellet.
next_step();
dry_pellet(eppendorf2, "in air");
// 17. Dissolve each in 50μL TE OR H2O.
next_step();
first_option();
measure_fluid(te, vol(50, UL), eppendorf2);
next_option();
measure_fluid(water, vol(50, UL), eppendorf2);
end_option();
dissolve(eppendorf2);
end_protocol();
}