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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
| Step | Condition | Time |
| 1 | 95℃ | 5min |
| 2 | 95℃ | 30-50sec |
| 3 | {Tm(fu)+[(-2)~5]}℃ | 40-80sec |
| 4 | DNA length/kb/min | 10uM |
| 5 | RETURN TO STEP 2 | 10-15 cycles |
| 6 | 72℃ | 5min |
| 7 | Add amplification Primers | |
| 8 | 95℃ | 2-5min |
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
BioBrickTM Parts Making Protocol 1. get desired sequences through NCBI or other sources and check for restriction sites For no (Xba1 or Spe1) Goto STEP2 Else Goto STEP9 2. Design primers with half-prefix (Xba1) and half-suffix (Spe1) 3. PCR from according genome/plasmid 4. Purify PCR product using Gel Extraction Kit (Transgen) 5. Digest with Xba1+Spe1 6. Ligation with pSB1AC3, which was digest with Xba1+Spe1 and treated with CIAP 7. Transform to TOP10 cells 8. Identify clones with colony PCR Goto STEP20 9. Design primers with full-prefix and full-suffix 10. PCR from according genome/plasmid 11. Purify PCR product using Gel Extraction Kit (Transgen) For EcoR1 Goto STEP12 Else Goto STEP14 12. Digest with Xba1+Pst1 13. Ligation with pSB1AC3, which was digest with Xba1+Pst1 and treated with CIAP Goto STEP16 14. Digest with EcoR1+Spe1 15. Ligation with pSB1AC3, which was digest with EcoR1+Spe1 and treated with CIAP 16. Transform to TOP10 cells 17. Identify clones with colony PCR 18. Extract plasmid and site-directed mutate by fusion PCR 19. Transform to TOP10 cells 20. Extract plasmid and send sequencing End^^67
- Click on any day below to see what wet-lab procedures were conducted.
