Template:SJTU-BioX-Shanghai/Notebook/Construction/w8
Construction
Aug. 25
PCR cloning of fragment Pdark-GFP and gel extraction. Final concentration: 119.5ng/μl (tube1); 304ng/μl (tube2)
Aug. 26
Linearization of pSB1C3 plasmid by PCR.
| BBa_J0450 | |
|---|---|
| ddwater | 160μl |
| Template | 32μl |
| Primer pSB-F(10μM) | 8μl |
| Primer pSB-R(10μM) | 8μl |
| PrimeSTAR Mix | 200μl |
| Total volume | 400μl(50μl*8) |
DpnI digestion for 2h. Gel extraction.
Shipping part Pdark-GFP, Pdark-HR and PcpcG2-HR into pSB1C3 plasmid via PCR - Homologous recombination method (24 tubes).
| Parts | Control-1(*3) | Control-2(*3) | |
|---|---|---|---|
| ddwater | 300μl | 14μl | 15μl |
| 5x CEII Buffer | 100μl | 4μl | 4μl |
| Linearized plasmid pSB1C3 | 25μl | 1μl | 0μl |
| Fragment (Pdark-GFP/HR,PcpcG2-HR) | 25μl | 1μl | 1μl |
| Exnase II | 50μl | 0μl | 2μl |
Agarose Gel electrophoresis test.
Aug. 27
Transformation into DH5a (4 tubes not including no-fragment control) and plating afterwards.
* (Lack of control group led to a failure of this transformation.)
Test with colony PCR and restriction enzyme reaction (XbaI and PstI).
Plasmid extraction (Pdark-GFP).
Aug. 28
Linearization of official vector plasmid pSB1C3.
| BBa_J0450 | |
|---|---|
| ddwater | 160μl |
| Template | 32μl |
| Primer pSB-F(10μM) | 8μl |
| Primer pSB-R(10μM) | 8μl |
| PrimeSTAR Mix | 200μl |
| Total volume | 400μl(50μl*8) |
DpnI digestion for 2h. Gel extraction.
PCR for fragment PcpcG2-HR.
| PcpcG2-HR | Pdark-HR | |
|---|---|---|
| ddwater | 220μl | 220μl |
| Template | 10μl | 10μl |
| Primer UHKD-F(10μM) | 10μl | 10μl |
| Primer UHKD-R(10μM) | 10μl | 10μl |
| PrimeSTAR Mix | 250μl | 250μl |
| Total volume | 500μl(50μl*8) | 500μl(50μl*8) |
Agarose gel electrophoresis test and gel extraction for Pdark-HR and PcpcG2-HR fragment.
Shipping part Pdark-HR and PcpcG2-HR into pSB1C3 plasmid via PCR - Homologous recombination method.
Second try of homologous recombination.
| Pdark-HR | PcpcG2-HR | Non-fragment control | |
|---|---|---|---|
| ddwater | 12μl | 12μl | 13μl |
| 5x CEII Buffer | 4μl | 4μl | 4μl |
| Linearized plasmid pSB1C3 (80ng/μl) | 1μl | 1μl | 1μl |
| Fragment | 1μl | 1μl | 0μl |
| Exnase II | 2μl | 2μl | 2μl |
Transformation into DH5a (3 tubes) and plating afterwards.
Spread plates and cultured for 14h in 37℃.
Aug. 29
Inoculation of Pdark-GFP, Pdark-HR and PcpcG2-HR transformed DH5a. (11 tubes each, 3 from control group)
Aug. 30
Bacteria Liquid PCR fpr Pdark-GFP, Pdark-HR and PcpcG2-HR. Agarose gel electrophoresis test.
Pdark-GFP:
| pSB-vF | 1μl (10μM) |
| GFP-R | 1μl (10μM) |
| Bacterial liquid(Pdark-GFP) | 1μl |
| 2×Taq Mix | 10μl |
| ddwater | Fill up to 20μl |
| Total volume | 20μl |
| 95℃ 3min | |
| 94℃ 15s | ×28 cycle |
| 48℃ 15s | |
| 72℃ 1min20s | |
| 72℃ 5min | |
| 12℃ ∞ |
Pdark-HR:
| pSB-vF | 1μl (10μM) |
| SS-HR-R | 1μl (10μM) |
| Bacterial liquid(Pdark-GFP) | 1μl |
| 2×Taq Mix | 10μl |
| ddwater | Fill up to 20μl |
| Total volume | 20μl |
| 95℃ 3min | |
| 94℃ 15s | ×28 cycle |
| 48℃ 15s | |
| 72℃ 50s | |
| 72℃ 5min | |
| 12℃ ∞ |
PcpcG2-HR:
| pSB-vF | 1μl (10μM) |
| SS-HR-R | 1μl (10μM) |
| Bacterial liquid(Pdark-GFP) | 1μl |
| 2×Taq Mix | 10μl |
| ddwater | Fill up to 20μl |
| Total volume | 20μl |
| 95℃ 3min | |
| 94℃ 15s | ×28 cycle |
| 48℃ 15s | |
| 72℃ 60s | |
| 72℃ 5min | |
| 12℃ ∞ |