Silver:Restriction digest protocol - source code

#include "BioCoder.h"

void main()
{
	start_protocol("Silver- Restriction Digest");

	Fluid enzyme1 = new_fluid ("restriction enzyme1", "20 units/µl");
	Fluid enzyme2 = new_fluid ("restriction enzyme2", "20 units/µl");
	Fluid buffer = new_fluid ("10X restriction buffer");
	Fluid bsa = new_fluid ("10X BSA");
	Fluid dna = new_fluid("PCR product DNA");
	Fluid water = new_fluid("distilled H2O");
	Fluid vector = new_fluid("BioBrick vector", "BBa_V0002 or BBa_V0100");
	Fluid cip = new_fluid("CIP", "10 units/µl"); 

	Container rxn_tube1 = new_container(RXN_TUBE);
	Container rxn_tube2 = new_container(RXN_TUBE);

	Symbol v = new_symbol("V", "Volume of PCR product DNA obtained (µl).");
	Symbol c = new_symbol("C", "Concentration of BioBrick vector (µg/µl).");

	//#  Mix:
	//
	//    * All of PCR product DNA (~28 µL if PCR purification was eluted in 30 µL)
	//    * 3.5 µL 10x BSA
	//    * 3.5 µL 10x buffer (see NEB website for optimal double digest buffer choices)
	//    * 0.2 µL enzyme 1 (20 units/µL)
	//    * 0.2 µL enzyme 2 (20 units/µL)
	//    * distilled water to 35 µL total volume
	//    * Note: keep the glycerol concentration below 5%, the volume of both restriction enzymes added should not exceed 5% of the total reaction volume. 

	first_step("Digestion of PCR product");
	{
		Fluid fluid_array[6] = {dna, bsa, buffer, enzyme1, enzyme2, water};
		char* tubes[1] = {"Restriction Digest - PCR product"};
		Volume* volume[6] = {s_vol(v), vol(3.5, UL), vol(3.5, UL), vol(0.2, UL), vol(0.2, UL), vol(XVAL, UL)};
		mixing_table(2, 7, fluid_array, tubes, volume, vol(35, UL), rxn_tube1);
	}
	comment("Note: keep the glycerol concentration below 5%, the volume of both restriction enzymes added should not exceed 5% of the total reaction volume.");

	//# Incubate at least an hour (better overnight) at 37 °C.
	next_step();
	incubate(rxn_tube1, 37, min_time(1, HRS));
	comment("Incubation overnight is better.");

	//# Purify digested insert using PCR product purification kit
	next_step();
	comment("Purify digested insert using PCR product purification kit.");

	next_step("Digestion of BioBrick vector: Recommended procedure");
	/* 1.  Mix:
	* 700 ng BioBrick vector (BBa_V0002 or BBa_V0100)
	* 1 µL 10 x BSA
	* 1 µL 10x buffer (see NEB website for optimal double digest buffer choices)
	* 0.2 µL enzyme 1 (20 units/µL)
	* 0.2 µL enzyme 2 (20 units/µL)
	* distilled water to 10 µL total volume
	* Note: To keep the glycerol concentration below 5%, the volume of both restriction enzymes added should not exceed 10% of the total reaction volume. */
	{
		Fluid fluid_array[6] = {vector, bsa, buffer, enzyme1, enzyme2, water};
		char* tubes[1] = {"Restriction Digest - vector"};
		Volume* volume[6] = {s_vol(divide(vol(7, UL), s_vol(c))), vol(1, UL), vol(1, UL), vol(0.2, UL), vol(0.2, UL), vol(XVAL, UL)};
		mixing_table(2, 7, fluid_array, tubes, volume, vol(10, UL), rxn_tube2);
	}
	comment("Note: keep the glycerol concentration below 5%, the volume of both restriction enzymes added should not exceed 5% of the total reaction volume.");

	//2. Incubate overnight at 37 °C.
	next_step();
	incubate(rxn_tube1, 37, time(12, HRS));

	//3. The next morning, add 0.1 µL CIP (10 units/µL) and incubate for 1 hr. at 37 °C. 
	next_step();
	measure_fluid(cip, vol(0.1, UL), rxn_tube2);
	incubate(rxn_tube2, 37, time(1, HRS));


	end_protocol();
}