Toggle navigation SCU-Igem Project Background Overview Description Result Modeling Human Practice Presentation at Seventh Senior High School Visiting University of Electronic Science and Technology of China (UESTC) Safety Parts Attributions Team Notebook Notebook Notebook of Biobricks Notebook of Transmitter Notebook of Effector Notebook of Sensor Method Bacterial Genomic DNA Prep Digestion Gel Extraction Linkage PCR Plasmid Mini Prep PCR Protocol Back to top For constructing the Biobricks that we want to submit, we set up the PCR protocol and PCR system as bellows: PCR system (For 50μL) Materials Volume Taq DNA polymerase 0.5μL Template 0.5μL Forward Primer 1μL Reverse Primer 1μL dNTPs (2.5mM) 4μL 10x Buffer (Mg2+ free) 5μL MgCl2 (25mM) 3μL Sterilized diluted water 35μL Note: The content of template should be less than 500ng for the 50μL system. For all of our experiments, the content of template of 0.5μL DNA lower than 500ng. PCR protocolTemperature Times 95℃ 5min. 30-35 Times 95℃ 30sec. 55-65℃ 30sec. 72℃ 1-2min. 72℃ 10min. Note: for finding the optimal annealing temperature, we have done a series of gradient of annealing temperature first. And as for the elongation time, Taq DNA polymerase could synthesize DNA as the speed of 1000-2000 nucleotides per minute. Sichuan university
PCR Protocol
For constructing the Biobricks that we want to submit, we set up the PCR protocol and PCR system as bellows:
Materials
Volume
Taq DNA polymerase
0.5μL
Template
Forward Primer
1μL
Reverse Primer
dNTPs (2.5mM)
4μL
10x Buffer (Mg2+ free)
5μL
MgCl2 (25mM)
3μL
Sterilized diluted water
35μL
Note: The content of template should be less than 500ng for the 50μL system. For all of our experiments, the content of template of 0.5μL DNA lower than 500ng.
Temperature
Times
95℃
5min.
30sec.
55-65℃
72℃
1-2min.
10min.
Note: for finding the optimal annealing temperature, we have done a series of gradient of annealing temperature first. And as for the elongation time, Taq DNA polymerase could synthesize DNA as the speed of 1000-2000 nucleotides per minute.
Sichuan university
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