Endy:Colony PCR protocol - source code

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#include "BioCoder.h"
#define X 60

void main()
{
	start_protocol("Endy - Colony PCR");

	Fluid buffer = new_fluid("Thermo polymerase buffer");
	Fluid dntps = new_fluid("dNTPS");
	Fluid f_primer = new_fluid("Forward primer");
	Fluid r_primer = new_fluid("Reverse primer");
	Fluid taq = new_fluid("Taq or Vent Polymerase");
	Fluid temp_sus = new_fluid("Template suspension");
	Fluid h2o = new_fluid("H<sub>2</sub>O");

	Container rxn_tube = new_container(RXN_TUBE);

	first_step();
	comment("Use a sterile toothpick or pipet tip to resuspend a plated colony in 50 μl sterile water.");

	next_step();
	comment("Store the colony resuspension at 4°C so you can start cultures if necessary (should be OK for a couple days, if you need it to last longer you should use an Index plate.");
	// Reaction Mix
	//
	//Use the following reaction mix for each PCR:
	//
	//    * 1 μl 10x Thermo polymerase buffer
	//    * 1 μl 10x dNTPs (10x = 2.5 mM each dNTP)
	//    * 0.15 μl 40 μM FWD primer
	//    * 0.15 μl 40 μM REV primer
	//    * 0.1 μl Polymerase (taq or vent)
	//    * 6.6 μl H2O
	//    * 1.0 μl template suspension 
	next_step("Reaction mix");
	{
		Fluid fluid_array[7] = {buffer, dntps, f_primer, r_primer, taq, temp_sus, h2o};
		char* (initial_conc)[7] = {"10X", "10X", "40 µM", "40 µM", "--", "--", "--"};
		char* (final_conc)[7] = {"1X", "1X", "0.6 µM", "0.6 µM", "--", "--", "--"};
		Volume* volume[7] = {vol(1, UL), vol(1, UL), vol(0.15, UL), vol(0.15, UL), vol(0.1, UL), vol(6.6, UL), vol(1, UL)};
		mixing_table_pcr(8, vol(10, UL), fluid_array, initial_conc, final_conc, volume, rxn_tube);
	}

	//
	//PCR protocol
	//
	//    * 95 C for 6 minutes (disrupt cells, separate DNA)
	//    * Cycle 35 times:
	//          o 95 C for 30 s (melting)
	//          o 53 C (or whatever temperature is appropriate) for 30 s (annealing)
	//          o 72 C for X s (elongation) 
	//    * 72 C for 10 minutes (final elongation)
	//    * 4 C forever
	//    * For long amplicons, X = 1 minute + 2.5 s per 100bp
	//    * For shorter amplicons, under ~1kb, this can be shortened judiciously. 
	next_step("PCR protocol");
	pcr_init_denat(rxn_tube, 95, time(6, MINS));
	thermocycler(rxn_tube, 35, 95, time(30, SECS), 53, time(30, SECS), 72, time(X, SECS), NORMAL);
	pcr_final_ext(rxn_tube, 72, time(10, MINS), 4);
	comment("For long amplicons, elongation time = 1 minute + 2.5 s per 100bp.");
	comment("For shorter amplicons, under ~1kb, this can be shortened judiciously.");
	end_protocol();
}
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