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Team:Tsinghua/Notebook
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Basic Wet-lab protocols
PCR Fusion PCR Restriction cut Ligation Transformation
1. PCR
| Reagent | Concentration/Activity | 50ul | 100ul |
| 10xPyrobest bufferII | 10x | 5 | 10 |
| Pyrobest | 0.3 | 0.5 | |
| dNTPmix | 10mM each | 1 | 2 |
| Primer 1 | 10uM | 1 | 2 |
| Primer 2 | 10um | 1 | 2 |
| Template DNA | changeable | 0.5 | 1 |
| MgCl2(Deletable) | 0.2M | 0.5 | 1 |
| ddH2O | 40.5 | 81 |
2. Fusion PCR
2.1 System
The basic system is similar to common PCR. There are some notes to raise the fusion efficiency.
a. Complementary region length: 15-20bp
b. Raise the annealing temperature in the fusion step.
2.2 Program: 2.2.1 95℃ 5min
2.2.2 95℃ 30-50sec
2.2.3 {Tm(fu)+[(-2)~5]}℃ 40-80sec
2.2.4 72℃ DNA length/kb/min
2.2.5 return to 2.2.2 for 10-15 cycles
2.2.6 72℃ 5min
2.2.7 Add amplification Primers
2.2.8 95℃ 2-5min
2.2.9 continue common program for 25-30 cycles
3. Restriction Digestion
| Reagent | Concentration/Activity | Volume(50ul system) |
| Restriction cut buffer | 10x | 5ul |
| Enzyme 1 | 1ul | |
| Enzyme 2 | 1ul |
Add DNA and distilled water to 50ul.Incubate at 37℃, 1.5 hrs or longer(Enzymes from Takara Co., Ltd or NEB).
4. Ligation
| Reagent | Volume(10ul system) |
| Solution I | 5ul |
| DNA fragment | 3.5ul(changeable) |
| Vector | 1.5ul(changeable) |
Incubate at 16-18℃,1hr or longer(Ligation kit from Takara.,Ltd).
Notes: Advanced protocol for parts extraction
- Click on any day below to see what wet-lab procedures were conducted.
