Kafatos:Minipreps protocol - source code
From OpenWetWare
#include "BioCoder.h"
void main()
{
start_protocol("Katafos - Minipreps");
Fluid lb = new_fluid("5 ml sterile LB medium (+ antibiotic)");
Fluid p1 = new_fluid("P1 Buffer");
Fluid p2 = new_fluid("P2 Buffer");
Fluid p3 = new_fluid("P3 Buffer", ICE_COLD);
Fluid etoh = new_fluid("absolute EtOH");
Fluid etoh70 = new_fluid("70% ethanol");
Fluid te = new_fluid("TE");
Fluid water = new_fluid("ddH<sub>2</sub>O");
Solid colony = new_solid("single bacterial colony");
Container flask = new_container(FLASK);
Container eppendorf1 = new_container(EPPENDORF);
Container eppendorf2 = new_container(EPPENDORF);
//Day 1
//
//- inoculate 5 ml sterile LB medium (+ antibiotic ?) with single bacterial colony
//- grow ON @ 37˚C while shaking vigorously
first_step("DAY 1");
set_container(lb, flask);
inoculation(flask, colony, 37, time(12, HRS), 1);
//[edit] Day 2
//
//- spin 1.5 ml 2’ @ 8,000 rpm in table top centrifuge
next_step("DAY 2");
first_sub_step();
measure_fluid(flask, vol(1.5, ML), eppendorf1);
centrifuge_pellet(eppendorf1, speed(8000, RPM), RT, time(2, MINS));
//- resuspend pellet in 100 μl P1 Buffer. Let sit 5’ @ RT
next_sub_step();
measure_fluid(p1, vol(100, UL), eppendorf1);
resuspend(eppendorf1);
store_for(eppendorf1, RT, time(5, MINS));
//- add 200 μl P2 Buffer, mix and let sit no more than 5’ @ RT
next_sub_step();
measure_fluid(p2, vol(200, UL), eppendorf1);
store_for(eppendorf1, RT, max_time(5, MINS));
//- add 150 μl ice cold P3 Buffer, mix well and place 5 – 10’ on ice
next_sub_step();
measure_fluid(p3, vol(150, UL), eppendorf1);
vortex(eppendorf1);
store_for(eppendorf1, ON_ICE, time_range(5, 10, MINS));
//- spin 10’ @ max rpm (4˚C if possible)
next_sub_step();
centrifuge_phases(eppendorf1, speed(SPEED_MAX, RPM), 4, time(10, MINS), eppendorf2);
//- transfer supernatant to new tube and add 2 volumes of abs EtOH. Let sit for 10 – 20’ on ice
next_sub_step();
measure_prop_and_add(eppendorf2, etoh, 2);
store_for(eppendorf2, ON_ICE, time_range(10, 20, MINS));
//- spin 10’ @ max. speed @ 4˚C
next_sub_step();
centrifuge_pellet(eppendorf2, speed(SPEED_MAX, RPM), 4, time(10, MINS));
//- discard supernatant and wash pellet with 1ml of 70% EtOH
next_sub_step();
measure_fluid(etoh70, vol(1, ML), eppendorf2);
pipet(eppendorf2);
//- spin 5’ @ max speed
next_sub_step();
centrifuge_pellet(eppendorf2, speed(SPEED_MAX, RPM), RT, time(5, MINS));
//- suck up or decant supernatant, speedvac or dry pellet @ 37˚C (do not overdo it, though, or it will not properly dissolve again)
next_step();
first_option();
dry_pellet(eppendorf2, "in air");
next_option();
dry_pellet(eppendorf2, "in speedvac");
end_option();
comment("Do not overdo drying, or the pellet will not properly dissolve again.");
//- resuspend pellet in 30 μl TE Buffer or ddH20 and check on gel
next_step();
first_option();
measure_fluid(te, vol(30, UL), eppendorf2);
next_option();
measure_fluid(water, vol(30, UL), eppendorf2);
end_option();
resuspend(eppendorf2);
electrophoresis(eppendorf2);
end_protocol();
}