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

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:

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/Pst1
• digesting J23078 with Pst1/Xba1
• Digestion system contains:

1. R0040:2uL 10*H, 0.5uL SpeI, 0.5uL PstI, 10uL plasmid, 7uL ddH2O.
2. 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.

1. Result: In Taq PCR all 6 pairs of primers show bright bands at 500 bp, while none is shown in Pfu PCR.
2. 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.