Mouse tissue lysis for genotyping protocol - source code

From OpenWetWare
Jump to navigationJump to search

#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 
	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);