Blackburn:Yeast Colony PCR v2.0 protocol - source code: Difference between revisions
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<code><pre> | <code><pre> | ||
#include "BioCoder.h" | #include "BioCoder.h" | ||
#define X 60 | |||
void main() | void main() | ||
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Fluid water = new_fluid("ddH<sub>2</sub>O"); | Fluid water = new_fluid("ddH<sub>2</sub>O"); | ||
Solid colony = new_solid("a small colony"); | Solid colony = new_solid("a small colony"); | ||
Container pcr_tube1 = new_container(STERILE_PCR_TUBE); | Container pcr_tube1 = new_container(STERILE_PCR_TUBE); | ||
Line 83: | Line 82: | ||
next_sub_step(); | next_sub_step(); | ||
pcr_init_denat(pcr_tube3, 95, time(5, MINS)); | pcr_init_denat(pcr_tube3, 95, time(5, MINS)); | ||
thermocycler(pcr_tube3, 30, 95, time(10, SECS), 50, time(10, SECS), 72, time( | thermocycler(pcr_tube3, 30, 95, time(10, SECS), 50, time(10, SECS), 72, time(X, SECS), NORMAL); | ||
comment("I generally do 30 sec elongation."); | comment("Elongation time : 1 min/kbp.I generally do 30 sec elongation."); | ||
pcr_final_ext(pcr_tube3, 72, time(10, MINS), 4); | pcr_final_ext(pcr_tube3, 72, time(10, MINS), 4); | ||
comment("I don't think this step is critical."); | comment("I don't think this step is critical."); |
Latest revision as of 00:05, 20 November 2009
#include "BioCoder.h"
#define X 60
void main()
{
start_protocol("Blackburn - Yeast Colony PCR");
Fluid naoh = new_fluid("0.02M NaOH");
Fluid q_solution = new_fluid("Q-solution", "5X");
Fluid pcr_buffer = new_fluid("PCR buffer", "10X");
Fluid dntp = new_fluid("dNTPs", "10mM each");
Fluid f_primer = new_fluid("Forward primer", "100µM");
Fluid r_primer = new_fluid("Reverse primer");
Fluid taq = new_fluid("Taq");
Fluid water = new_fluid("ddH<sub>2</sub>O");
Solid colony = new_solid("a small colony");
Container pcr_tube1 = new_container(STERILE_PCR_TUBE);
Container pcr_tube2 = new_container(STERILE_PCR_TUBE);
Container pcr_tube3 = new_container(STERILE_PCR_TUBE);
//Yeast Cell Lysis
// 1. Aliquot 10uL of 0.02M NaOH into PCR tubes.
first_step("Yeast Cell Lysis");
first_sub_step();
measure_fluid(naoh, vol(10, UL), pcr_tube1);
// 2. Using a sterile pipette tip, pick a small colony and resuspend in NaOH.
// * If the solution is cloudy, you've added enough cells.
// * I have been told adding too much yeast can inhibit the reaction.
next_sub_step();
measure_solid(colony, pcr_tube1);
name_sample(pcr_tube1, "colony");
resuspend(pcr_tube1);
comment("If the solution is cloudy, you've added enough cells.");
comment("I have been told adding too much yeast can inhibit the reaction.");
// 3. Boil the samples on a PCR machine by incubating the tubes at 99C for 10 minutes.
// * In the mean time, prepare the master mix for the PCR reaction.
// * The boiled samples are stable at room temp for some time. Keep on ice or freeze for longer.
next_sub_step();
thermocycler(pcr_tube1, 99, time(10, MINS));
comment("In the mean time, prepare the master mix for the PCR reaction.");
comment("The boiled samples are stable at room temp for some time. Keep on ice or freeze for longer.");
//PCR
// 1. Prepare the master mix solution containing:
// * 2uL 5X Q-solutioCn
// * 1uL 10X PCR Buffer
// * 0.2uL dNTPs (10mM each)
// * 0.2uL foward primer (100uM)
// * 0.2uL reverse primer (100uM)
// * 0.1uL Taq
// * 5.3uL ddH2O
next_step("PCR");
first_sub_step();
Fluid fluid_array[7] = {q_solution, pcr_buffer, dntp, f_primer, r_primer, taq, water};
Volume* volumes[7] = {vol(2, UL), vol(1, UL) , vol(0.2, UL), vol(0.2, UL), vol(0.2, UL), vol(0.1, UL), vol(5.3, UL)};
char* tube[1] = {"Colony PCR"};
mixing_table(2, 8, fluid_array, tube, volumes, vol(9, UL), pcr_tube2);
// 2. Aliquot 9uL of the master mix solution into fresh PCR tubes.
next_sub_step();
name_container(pcr_tube3, "Master Mix aliquot");
name_sample(pcr_tube2, "master mix solution");
measure_fluid(pcr_tube2, vol(9, UL), pcr_tube3);
// 3. Transfer 1uL of boiled samples to the master mix aliquots (a multi-channel pipette is helpful here).
next_sub_step();
name_sample(pcr_tube1, "boiled sample");
measure_fluid(pcr_tube1.contents, vol(1, UL), pcr_tube3);
comment("A multichannel pipette is helpful here.");
// 4. Run the following PCR cycle:
// 1. 5 min at 95C
// 2. 30 cycles of:
// 1. 10 sec at 95C
// 2. 10 sec at 50C (or appropriate annealing temperature)
// 3. 1 min/kbp at 72C (I generally do 30 sec)
// 3. 10 min at 72C (I don't think this step is critical)
next_sub_step();
pcr_init_denat(pcr_tube3, 95, time(5, MINS));
thermocycler(pcr_tube3, 30, 95, time(10, SECS), 50, time(10, SECS), 72, time(X, SECS), NORMAL);
comment("Elongation time : 1 min/kbp.I generally do 30 sec elongation.");
pcr_final_ext(pcr_tube3, 72, time(10, MINS), 4);
comment("I don't think this step is critical.");
end_protocol();
}