IGEM:Peking/2007/Count-Conjugation-Notebook/2007-7-23

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(Riboregulator)
(electrophorese the product of digsting test)
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1 e0040->pSB3k3-1+EcoR1 2 e0040->pSB3k3-2+EcoR1 3 e0040->pSB3k3-1+EcoR1+Pst1 4 e0040->pSB3k3-2+EcoR1+Pst1
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
5 marker: DL2000 plus 6 e0040->pSB3k3-2+plasmid 7 e0040->pSB3k3-1 plasmid
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[[Image:2007-7-23 EGFP-pSB3k3-1_EcoR1+Pst1 and EGFP-pSB3k3-2_EcoR1+Pst1.jpg]]
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===digesting R0040 and J23078(crRNA) for ligation===
===digesting R0040 and J23078(crRNA) for ligation===

Revision as of 22:46, 23 July 2007

Contents

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 Image:2007-7-23 EGFP-pSB3k3-1_EcoR1+Pst1 and EGFP-pSB3k3-2_EcoR1+Pst1.jpg

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.
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