IGEM:Peking/2007/Count-Conjugation-Notebook/2007-7-23: Difference between revisions
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===Riboregulator=== | ===Riboregulator=== | ||
===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 | |||
*for 2 hours | |||
===electrophorese the product of digsting test=== | |||
*from left to right: | |||
1 e0040->pSB3k3-1+EcoR1 2 e0040->pSB3k3-2+EcoR1 3 e0040->pSB3k3-1+EcoR1+Pst1 4 e0040->pSB3k3-2+EcoR1+Pst1 | |||
5 marker: DL2000 plus 6 e0040->pSB3k3-2+plasmid 7 e0040->pSB3k3-1 plasmid | |||
===digesting R0040 and J23078(crRNA) for ligation=== | |||
*digesting R0040 with Spe1 and Pst1 | |||
*digesting J23078 with Pst1 and Xba1 | |||
*for 15 hours | |||
===oriT Knock Out=== | ===oriT Knock Out=== | ||
Revision as of 19:43, 23 July 2007
Riboregulator
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
- for 2 hours
electrophorese the product of digsting test
- from left to right:
1 e0040->pSB3k3-1+EcoR1 2 e0040->pSB3k3-2+EcoR1 3 e0040->pSB3k3-1+EcoR1+Pst1 4 e0040->pSB3k3-2+EcoR1+Pst1 5 marker: DL2000 plus 6 e0040->pSB3k3-2+plasmid 7 e0040->pSB3k3-1 plasmid
digesting R0040 and J23078(crRNA) for ligation
- digesting R0040 with Spe1 and Pst1
- digesting J23078 with Pst1 and Xba1
- for 15 hours
oriT Knock Out
- By Xu Anting
- 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.
