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Team:Tsinghua/Notebook
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Basic Wet-lab protocols
PCR Fusion PCR Restriction cut Ligation Transformation
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 |
Fusion PCR
(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) Program:
• Program:
• 95’: 5min
• 95' : 30-50sec
• Tm(fu)+ (-2)~5: 40-80sec
• 72' : the longer/1kb/min 10-15 cycles • 72' 5min • Add amplification Primers • 95' 2-5min • Go on under common program for 25-30 cycles
Restriction cut
| 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).
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.
