Richard Lab:Restriction Digest protocol - source code

From OpenWetWare

(Difference between revisions)
Jump to: navigation, search
Current revision (00:46, 20 November 2009) (view source)
 
Line 6: Line 6:
start_protocol("Richard's Lab - Restriction Digestion");
start_protocol("Richard's Lab - Restriction Digestion");
-
Fluid dna = new_fluid("prepared DNA", "from Miniprep, PCR or Gel Extraction");
+
Fluid dna = new_fluid("prepared DNA", "from Miniprep, PCR or Gel Extraction", vol(20, UL));
Fluid re = new_fluid("restriction endonucleases");
Fluid re = new_fluid("restriction endonucleases");
Fluid re_buffer = new_fluid("10X restriction endonuclease buffer");
Fluid re_buffer = new_fluid("10X restriction endonuclease buffer");
Line 31: Line 31:
next_step();
next_step();
measure_fluid(dna, rxn_tube1);
measure_fluid(dna, rxn_tube1);
-
measure_prop_and_add(rxn_tube1, water, 4);
+
measure_prop(rxn_tube1, water, 4);
name_sample(rxn_tube1, "DNA solution");
name_sample(rxn_tube1, "DNA solution");
Fluid fluid_array[4] = {re_buffer, bsa, rxn_tube1.contents, re};
Fluid fluid_array[4] = {re_buffer, bsa, rxn_tube1.contents, re};
-
Volume* volumes[4] = {vol(5, UL), vol(1, UL), vol(40, UL), vol(1, UL)};
+
Volume* volumes[4] = {vol(5, UL), vol(1, UL), vol(40, UL), vol(4, UL)};
char* tubes[1] = {"Restriction Digestion"};
char* tubes[1] = {"Restriction Digestion"};
mixing_table(2, 5, fluid_array, tubes, volumes, vol(50, UL), rxn_tube2);
mixing_table(2, 5, fluid_array, tubes, volumes, vol(50, UL), rxn_tube2);
Line 47: Line 47:
store_for(rxn_tube2, 75, time(15, MINS), ENZYME_INAC);
store_for(rxn_tube2, 75, time(15, MINS), ENZYME_INAC);
-
//6. If digesting a vector add 10 units (1µl) phosphatase) and 5µl Phosphatase Buffer  
+
//6. If digesting a vector add 10 units (1µl) phosphatase) and 5µl Phosphatase Buffer and incubate an additional 45 minutes at then heat kill againat 75°C for 15 min.
-
        //and incubate an additional 45 minutes at then heat kill againat 75°C for 15 min.
+
next_step("If digesting vector:");
next_step("If digesting vector:");
measure_fluid(phosphatase, vol(1, UL), rxn_tube2);
measure_fluid(phosphatase, vol(1, UL), rxn_tube2);

Current revision

#include "BioCoder.h"

void main()
{
	start_protocol("Richard's Lab - Restriction Digestion");

	Fluid dna = new_fluid("prepared DNA", "from Miniprep, PCR or Gel Extraction", vol(20, UL));
	Fluid re = new_fluid("restriction endonucleases");
	Fluid re_buffer = new_fluid("10X restriction endonuclease buffer");
	Fluid bsa = new_fluid("BSA", "optional");
	Fluid phosphatase = new_fluid("phosphatase");
	Fluid phosphatase_buff = new_fluid("phosphatase buffer");
	Fluid water = new_fluid("distilled water");

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

	//Procedure
	//
	//1. Quickly vortex all ingredients (Buffer, BSA, DNA, Enzymes) before beginning
	first_step();
	to_do("Quickly vortex all ingredients (Buffer, BSA, DNA, Enzymes) before beginning.");


	//2. Add the following in a micro-centrifuge tube
	//         1. 5μl of Buffer
	//         2. 1μl of BSA
	//         3. 40μl of DNA solution (Dilute PCR products 1:4) 
	//3. Vortex Enzymes and add 20 units (1μl) of each to the tube
	next_step();
	measure_fluid(dna, rxn_tube1);
	measure_prop(rxn_tube1, water, 4);
	name_sample(rxn_tube1, "DNA solution");
	Fluid fluid_array[4] = {re_buffer, bsa, rxn_tube1.contents, re};
	Volume* volumes[4] = {vol(5, UL), vol(1, UL), vol(40, UL), vol(4, UL)};
	char* tubes[1] = {"Restriction Digestion"};
	mixing_table(2, 5, fluid_array, tubes, volumes, vol(50, UL), rxn_tube2);

	//4. Incubate reaction in a 37°C water bath for at least one hour (I like 1:45).
	next_step();
	incubate(rxn_tube2, 37, min_time(1, HRS));
	comment("Use a water bath for incubation.");

	//5. Heat kill the digest for 15 minutes at 75°C.
	next_step();
	store_for(rxn_tube2, 75, time(15, MINS), ENZYME_INAC);

	//6. If digesting a vector add 10 units (1µl) phosphatase) and 5µl Phosphatase Buffer and incubate an additional 45 minutes at then heat kill againat 75°C for 15 min.
	next_step("If digesting vector:");
	measure_fluid(phosphatase, vol(1, UL), rxn_tube2);
	measure_fluid(phosphatase_buff, vol(5, UL), rxn_tube2);
	incubate(rxn_tube2, 37, min_time(45, MINS));
	store_for(rxn_tube2, 75, time(15, MINS), ENZYME_INAC);

	//7. Store digested DNA in the freezer (-20°C). 
	next_step();
	store(rxn_tube2, -20);

	end_protocol();
}
Personal tools