Chromosomal DNA isolation from E. coli protocol - source code
From OpenWetWare
#include "BioCoder.h"
void main()
{
start_protocol("Chromosomal DNA isolation from E.coli");
Fluid culture = new_fluid("culture grown in your favorite medium");
Fluid killing_buffer = new_fluid("Killing Buffer", ICE_COLD);
Fluid te = new_fluid("TE");
Fluid sds = new_fluid("10% SDS");
Fluid edta = new_fluid("0.5M EDTA");
Fluid isopropanol = new_fluid("isopropanol");
Fluid rnase = new_fluid("RNase A", "25mg/ml");
Fluid proteinasek = new_fluid("proteinase K", "25mg/ml");
Fluid ethanol = new_fluid("ethanol");
Fluid naac = new_fluid("3M Na-Acetate");
Fluid etoh70 = new_fluid("70% ethanol");
Fluid water = new_fluid("distilled water");
Container eppendorf1 = new_container(EPPENDORF);
Container eppendorf2 = new_container(EPPENDORF);
// * grow culture in your favorite medium
// * Mix samples directly with ice cold Killing Buffer in ratio 1:1 and put on ice (samples should be processed as fast as possible)
first_step();
measure_fluid(culture, eppendorf1);
measure_prop_and_add(eppendorf1, killing_buffer, 1);
vortex(eppendorf1);
store(eppendorf1, ON_ICE);
comment("Samples should be processed as fast as possible.");
// * Spin down cells 3 min max. speed at 4°C and discard supernatant
next_step();
centrifuge_pellet(eppendorf1, speed(SPEED_MAX, RPM), 4, time(3, MINS));
// * resuspend in 300μL TE and add 40μL 10%SDS and 3μL 0.5M EDTA
next_step();
measure_and_add(eppendorf1, te, vol(300, UL));
measure_and_add(eppendorf1, sds, vol(40, UL));
measure_and_add(eppendorf1, edta, vol(3, UL));
resuspend(eppendorf1);
// * incubate 5 min at 65°C
next_step();
incubate(eppendorf1, 65, time(5, MINS));
// * add 750μL isopropanole and mix
next_step();
measure_and_add(eppendorf1, isopropanol, vol(750, UL));
vortex(eppendorf1);
// * spin at max. speed for 5 min
next_step();
centrifuge_pellet(eppendorf1, speed(SPEED_MAX, RPM), RT, time(5, MINS));
// * resuspend pellet in 500μL TE and add 2μL RNase A (25mg/ml)
next_step();
measure_and_add(eppendorf1, te, vol(500, UL));
measure_and_add(eppendorf1, rnase, vol(2, UL));
resuspend(eppendorf1);
// * incubate for 30 min at 65°C
next_step();
incubate(eppendorf1, 65, time(30, MINS));
// * add 2μL proteinase K (25mg/ml) and incubate at 37°C for 15 min
next_step();
measure_and_add(eppendorf1, proteinasek, vol(2, UL));
incubate(eppendorf1, 37, time(15, MINS));
// * phenol extract (2x phenol & 2x chlorophorm)
next_step();
to_do("phenol extract (2x phenol & 2x chlorophorm).");
// * precipitate over night with 1ml ethanol and 40μL 3M Na-Acetate
next_step();
measure_and_add(eppendorf1, ethanol, vol(1, ML));
measure_and_add(eppendorf1, naac, vol(40, UL));
store_for(eppendorf1, RT, time(12, HRS));
// * spin down DNA at 4°C for 15 min, wash in 70% ethanol and resuspend pellet in 50μL dH2O
next_step();
centrifuge_pellet(eppendorf1, speed(SPEED_MAX, RPM), 4, time(15, MINS));
measure_and_add(eppendorf1, water, vol(50, UL));
resuspend(eppendorf1);
end_protocol();
}