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.