Template:SJTU-BioX-Shanghai/Notebook/Construction/w1
Construction
July 7
1. Get promotor PcpcB fragment and Kn (kanamycin) fragment respectively from plasmid Bluescript and PRSF by PCR
| PcpcB (from pBluescript) | Kn (from pRSF) | |
|---|---|---|
| ddwater | 45.5μl | 45.5μl |
| Template | 0.5μl(1ng/50μl) | 0.5μl(1ng/50μl) |
| Primer1(10μM) | 2μl | 2μl |
| Primer2(10μM) | 2μl | 2μl |
| PrimeSTAR Mix | 50μl | 50μl |
| Total volume | 100μl (50μl*2) | 100μl (50μl*2) |
| ×34 cycle | 98℃ 3min | 98℃ 3min |
| 98℃ 10s | 98℃ 10s | |
| 56℃ 5s | 58℃ 5s | |
| 72℃ 3s | 72℃ 5s | |
| 72℃ 3min | 72℃ 3min | |
| 12℃ ∞ | 12℃ ∞ | |
| Final concentration(after purification) | 165ng/μl | 225ng/μl |
Gel analyses and purification.
2. Link aforementioned fragments by overlapping PCR to acquire fragment PcpcB-Kn
| ddwater | 90μl |
| Template1(PcpcB) | 1.2μl(50~100ng/50μl) |
| Template2(Kn) | 1.44μl(50~100ng/50μl) |
| Primer1(10μM) | 4μl |
| Primer2(10μM) | 4μl |
| PrimeSTAR mix | 100μl |
| Total volume | 200μl (50μl*4) |
| (round 1)×8 cycle | 98℃ 3min |
| 98℃ 10s | |
| 53℃ 5s | |
| 72℃ 4s | |
| 72℃ 3min | |
| 12℃ ∞ | |
| (round 2)×35 cycle | 98℃ 3min |
| 98℃ 10s | |
| 63℃ 5s | |
| 72℃ 7s | |
| 72℃ 3min | |
| 12℃ ∞ | |
| Final concentration(after purification) | Sample1+2: 35ng/μlSample3+4: 54ng/μl |
July 9
- Gel analyses for PcpcB-Kn.
- DNA purification to get purified fragment PcpcB-Kn.
July 10
- PCR to get fragment PcpcB-Kn-Down. Gel analyses for PcpcB-Kn-Down.
- Gel extraction to get purified fragment.
July 12
- Link PcpcB-Kn-Down and Up-GFP (had prepared) by overlapping PCR to get the entire sequence of P_dark-GFP and gel analyses.
No correct band is showed. - Transformation of E.coli to amplify pBluescript.