Mouse tissue lysis for genotyping protocol - source code: Difference between revisions
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(New page: <code> <pre> #include "BioCoder.h" void main() { start_protocol("Mouse tissue lysis for genotyping"); Fluid taq = new_fluid("Taq DNA polymerase 10X buffer with MgCl<sub>2</sub>", "dilu...) |
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Latest revision as of 04:36, 22 March 2010
#include "BioCoder.h"
void main()
{
start_protocol("Mouse tissue lysis for genotyping");
Fluid taq = new_fluid("Taq DNA polymerase 10X buffer with MgCl<sub>2</sub>", "diluted 1:10 in ddH<sub>2</sub>");
Fluid proteinasek = new_fluid("proteinase K", "20 mg/ml");
Solid tissue = new_solid("tail or tissue chunk");
Container tube1 = new_container(STERILE_MICROFUGE_TUBE, tissue);
Container tube2 = new_container(STERILE_MICROFUGE_TUBE);
//tissue lysis to release DNA
//per tail or tissue chunk add (tissue degradation is sped up if tissue/tail is cut into smaller bits):
//* 100 µl Taq DNA polymerase 10x Reaction buffer with MgCl2 (e.g. Promega #M1883) diluted 1:10 in ddH2O
//* +2 µl proteinase K (20 mg/ml)
first_step("Tissue lysis to release DNA");
measure_fluid(taq, vol(100, UL), tube1);
measure_fluid(proteinasek, vol(2, UL), tube1);
//* incubate for 3h to o/n at 50°C at 300 rpm
next_step();
incubate(tube1, 50, time_range(3, OVERNIGHT, HRS), 300);
comment("proteinase K is stable over a broad pH range (4.0 to 12.5, optimum pH 8.0) and is also stable over the temperature range of 25 to 65°C.");
//proteinase K inactivation
//* 95°C for 10-20 min at 300 rpm
//* centrifuge at 13000 rpm for 3 min and transfer supernatant (80 µl) into new eppi
next_step("Proteinase K inactivation");
incubate(tube1, 95, time_range(10, 20, MINS), 300);
centrifuge_phases_top(tube1, speed(13000, RPM), RT, time(3, MINS), vol(80, UL), tube2);
name_sample(tube2, "sample");
//storage before PCR
//* 4°C short term, -20°C long term
next_step("Storage before PCR");
use_or_store(tube2, time(1, HRS), 4, -20);
end_protocol();
}