IGEM:Peking/2007/Count-Conjugation-Notebook/2007-7-23: Difference between revisions
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===electrophorese the product of digsting test=== | ===electrophorese the product of digsting test=== | ||
*from left to right: | *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 | |||
[[Image:2007-7-23 EGFP-pSB3k3-1_EcoR1+Pst1 and EGFP-pSB3k3-2_EcoR1+Pst1.jpg]] | [[Image:2007-7-23 EGFP-pSB3k3-1_EcoR1+Pst1 and EGFP-pSB3k3-2_EcoR1+Pst1.jpg]] | ||
（下次建议在图片上直接标注号，不过如果你们有备案打印版本的话就没有问题了--by Qu) | |||
===digesting R0040 and J23078(crRNA) for ligation=== | ===digesting R0040 and J23078(crRNA) for ligation=== | ||
Revision as of 02:44, 24 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:
- 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
（下次建议在图片上直接标注号，不过如果你们有备案打印版本的话就没有问题了--by Qu)
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.
