IGEM:Peking/2007/Count-Conjugation-Notebook/2007-7-23: Difference between revisions

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Digested crRNA ( J23078 ) and R0040 for ligation tommorrow.
=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.  


===oriT Knock Out===
==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
[[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/Pst1
*digesting J23078 with Pst1/Xba1
*Digestion system contains:
#R0040:2uL 10*H, 0.5uL SpeI, 0.5uL PstI, 10uL plasmid, 7uL ddH2O.
#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
*By Xu Anting
:Use chromosome extraction product as template to do PCR, with Taq or Pfu added.
==PCR for oriT-Deleted Fragments==
::Result: In Taq PCR all 6 pairs of primers show bright bands at 500 bp, while none is shown in Pfu PCR.
*Use chromosome extraction product as template to do PCR, with Taq or Pfu added.
::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.
#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.

Latest revision as of 19:47, 15 August 2007

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.
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