Knight:Colony PCR protocol - source code - Revision history

Vaishnavi Ananth at 05:36, 20 November 2009

2009-11-20T05:36:19Z

←Older revision		Revision as of 05:36, 20 November 2009
Line 1:		Line 1:
-	<pre>	+	<code><pre>
	#include "BioCoder.h"		#include "BioCoder.h"
		+	#define X 60

	void main()		void main()
Line 13:		Line 14:

	Container rxn_tube = new_container(RXN_TUBE);		Container rxn_tube = new_container(RXN_TUBE);
-
-	~~Symbol x = new_parameter("X", "1 min per kb of expected product.");~~

	// Reaction mixture		// Reaction mixture
Line 29:		Line 28:
	Volume* volumes[4] = {vol(9, UL), vol(0.25, UL), vol(0.25, UL), vol(0.5, UL)};		Volume* volumes[4] = {vol(9, UL), vol(0.25, UL), vol(0.25, UL), vol(0.5, UL)};
	char* tube[1] = {"Colony PCR"};		char* tube[1] = {"Colony PCR"};
-	mixing_table(2, 5, fluid_array, tube, volumes, vol(10, UL), rxn_tube);	+	mixing_table(2, 5, fluid_array, tube, volumes, vol(10, UL), rxn_tube);

	//PCR conditions		//PCR conditions
Line 44:		Line 43:
	next_step("PCR conditions");		next_step("PCR conditions");
	pcr_init_denat(rxn_tube, 95, time(15, MINS));		pcr_init_denat(rxn_tube, 95, time(15, MINS));
-	thermocycler(rxn_tube, 39, 94, time(30, SECS), 56, time(30, SECS), 68, time(~~XVAL~~, ~~MINS~~), NORMAL);	+	thermocycler(rxn_tube, 39, 94, time(30, SECS), 56, time(30, SECS), 68, time(X, SECS), NORMAL);
-	comment("Elongation time : I typically round up for this step. i.e. For a 3.6kb construct, I used a 4 min elongation time. It seems to help to be a bit generous with the elongation time.");	+	comment("Elongation time : 1 min per kb of expected product. I typically round up for this step. i.e. For a 3.6kb construct, I used a 4 min elongation time. It seems to help to be a bit generous with the elongation time.");
	pcr_final_ext(rxn_tube, 60, time(20, MINS), 4);		pcr_final_ext(rxn_tube, 60, time(20, MINS), 4);

Line 55:		Line 54:
	end_protocol();		end_protocol();
	}		}
-	</pre>	+	</pre></code>

Vaishnavi Ananth at 09:38, 19 November 2009

2009-11-19T09:38:35Z

←Older revision		Revision as of 09:38, 19 November 2009
Line 1:		Line 1:
	<pre>		<pre>
-	#include "~~BioStream~~.h"	+	#include "BioCoder.h"

	void main()		void main()

Vaishnavi Ananth: New page:

 #include "BioStream.h"  void main() {  	start_protocol("Knight - Colony PCR");  	Fluid supermix = new_fluid("PCR supermix"); 	Fluid vf2 = new_fluid("VF2", "40 µM"); 	Fluid vr = new_...

2009-09-24T10:09:33Z

New page: <pre> #include "BioStream.h" void main() { start_protocol("Knight - Colony PCR"); Fluid supermix = new_fluid("PCR supermix"); Fluid vf2 = new_fluid("VF2", "40 µM"); Fluid vr = new_...

New page

<pre>
#include "BioStream.h"

void main()
{

start_protocol("Knight - Colony PCR");

Fluid supermix = new_fluid("PCR supermix");
Fluid vf2 = new_fluid("VF2", "40 µM");
Fluid vr = new_fluid("VR", "40 µM");
Fluid colony = new_fluid("colony template");

Container rxn_tube = new_container(RXN_TUBE);

Symbol x = new_parameter("X", "1 min per kb of expected product.");

// Reaction mixture
//
//1X Reaction
//
// * 9 μL PCR supermix
// * 0.25 μL 40μM VF2
// * 0.25 μL 40μM VR
// * 0.5 μL colony template
//
first_step("Reaction Mixture");
Fluid fluid_array[4] = {supermix, vf2, vr, colony};
Volume* volumes[4] = {vol(9, UL), vol(0.25, UL), vol(0.25, UL), vol(0.5, UL)};
char* tube[1] = {"Colony PCR"};
mixing_table(2, 5, fluid_array, tube, volumes, vol(10, UL), rxn_tube);

//PCR conditions
//
// 1. 95°C for 15 mins
// 2. 94°C for 30 secs
// 3. 56°C for 30 secs
// 4. 68°C for 1 min per kb of expected product
// * I typically round up for this step. i.e. For a 3.6kb construct, I used a 4 min elongation time. It seems to help to be a bit generous with the elongation time.
// 5. Repeat 2-4 39 times.
// 6. 68°C for 20 mins
// 7. 4°C forever
//
next_step("PCR conditions");
pcr_init_denat(rxn_tube, 95, time(15, MINS));
thermocycler(rxn_tube, 39, 94, time(30, SECS), 56, time(30, SECS), 68, time(XVAL, MINS), NORMAL);
comment("Elongation time : I typically round up for this step. i.e. For a 3.6kb construct, I used a 4 min elongation time. It seems to help to be a bit generous with the elongation time.");
pcr_final_ext(rxn_tube, 60, time(20, MINS), 4);

//Run a gel to determine amplification product length.
next_step();
name_sample(rxn_tube, "PCR products");
electrophoresis(rxn_tube);

end_protocol();
}
</pre>