"
Team:Tsinghua/Notebook
From 2008.igem.org
Ylzouyilong (Talk | contribs) |
Ylzouyilong (Talk | contribs) |
||
| Line 92: | Line 92: | ||
|align="right"|Volume(50ul system) | |align="right"|Volume(50ul system) | ||
|- | |- | ||
| - | |||
|Restriction cut buffer | |Restriction cut buffer | ||
|10x | |10x | ||
| Line 105: | Line 104: | ||
|align="right"|1ul | |align="right"|1ul | ||
|} | |} | ||
| - | Add DNA and distilled water to 50ul. | + | Add DNA and distilled water to 50ul.Incubate at 37℃, 1.5 hrs or longer(Enzymes from Takara Co., Ltd or NEB). |
| - | + | ||
| - | + | ||
Ligation | Ligation | ||
| - | + | {| border="1" | |
| - | + | |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: | Notes: | ||
Revision as of 17:57, 28 October 2008
| HOME | Team | Project | Parts | Modelling | Notebook | Doodle Board |
|---|
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.
