Knight:Colony PCR protocol - source code: Difference between revisions
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(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_...) |
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#include " | #include "BioCoder.h" | ||
void main() | void main() |
Revision as of 02:38, 19 November 2009
#include "BioCoder.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(); }