< IGEM:Peking | 2007 | Count-Conjugation-Notebook
Lock and Key By Zheng Qinsi and Yu Tao
digesting test of e0040->pSB3k3
- single digesting test of e0040->pSB3k3-1 and e0040->pSB3k3-2 with EcoR1
- double digesting test 0f e0040->pSB3k3-1 and e0040->pSB3k3-2 with EcoR1 and Pst1, respectively
- 37℃ culutre for 2 hours.
electrophorese the product of digsting test
from left to right:
- e0040->pSB3k3-1 @ EcoR1
- e0040->pSB3k3-2 @ EcoR1
- e0040->pSB3k3-1 @ EcoR1/Pst1
- e0040->pSB3k3-2 @ EcoR1/Pst1
- marker: DL2000 plus
- e0040->pSB3k3-2 plasmid
- e0040->pSB3k3-1 plasmid
digesting R0040 and J23078(crRNA) for ligation
- digesting R0040 with Spe1/Pst1
- digesting J23078 with Pst1/Xba1
- Digestion system contains:
- R0040:2uL 10*H, 0.5uL SpeI, 0.5uL PstI, 10uL plasmid, 7uL ddH2O.
- J23078:2uL 10*M, 0.5uL XbaI, 0.5uL PstI, 10uL plasmid, 7uL ddH2O.
- 37℃ culutre for 15 hours
OriT Knock Out
- By Xu Anting
PCR for oriT-Deleted Fragments
- Use chromosome extraction product as template to do PCR, with Taq or Pfu added.
- Result: In Taq PCR all 6 pairs of primers show bright bands at 500 bp, while none is shown in Pfu PCR.
- Conclusion: finally I got oriT up- and downstream fragments of 500 bp with potential site mutations caused by Taq's relatively low fidelity. I intend to use these products as template to do further PCR and get oriT-deleted fragments of 1200 bp.