IGEM:Peking/2007/Count-Conjugation-Notebook/2007-7-23: Difference between revisions
From OpenWetWare
Jump to navigationJump to search
Qinsi Zheng (talk | contribs) |
|||
| (5 intermediate revisions by 3 users not shown) | |||
| Line 1: | Line 1: | ||
== | =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 | *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 | *double digesting test 0f e0040->pSB3k3-1 and e0040->pSB3k3-2 with EcoR1 and Pst1, respectively | ||
*for 2 hours | *37℃ culutre 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 | |||
[[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]] | ||
==digesting R0040 and J23078(crRNA) for ligation== | |||
*digesting R0040 with Spe1 | *digesting R0040 with Spe1/Pst1 | ||
*digesting J23078 with Pst1 | *digesting J23078 with Pst1/Xba1 | ||
*for 15 hours | *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 | ||
==PCR for oriT-Deleted Fragments== | |||
*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. | |||
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:
- 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
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
PCR for oriT-Deleted Fragments
- 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.
