IGEM:Peking/2007/Count-Conjugation-Notebook/2007-7-17: Difference between revisions
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(New page: ===Miniprep=== *For E0040 and J23066; Note: pSB3K3 fails to grow in the preculture process. Possible reason: this pSB3K3 contains ccdB and the supposed transformants on the pSB3K3 plate a...) |
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===Miniprep | =Plasmid Construction= | ||
==Miniprep== | |||
*For E0040 and J23066; Note: pSB3K3 fails to grow in the preculture process. Possible reason: this pSB3K3 contains ccdB and the supposed transformants on the pSB3K3 plate are contaminated. | *For E0040 and J23066; Note: pSB3K3 fails to grow in the preculture process. Possible reason: this pSB3K3 contains ccdB and the supposed transformants on the pSB3K3 plate are contaminated. | ||
*Use Trans Kit. | *Use Trans Kit. | ||
*Final volume: 50uL for each plasmids. | *Final volume: 50uL for each plasmids. | ||
==Fetch the I7100== | |||
*We use I7100, which is inserted in pSB3K3, to substitute the pSB3K3 carrying the ccdB. | *We use I7100, which is inserted in pSB3K3, to substitute the pSB3K3 carrying the ccdB. | ||
==Digestion Test== | |||
*For pSB1AK3-1, R0040-1 and E0040-2 | *For pSB1AK3-1, R0040-1 and E0040-2 | ||
*EcoR I single digestion. | *EcoR I single digestion. | ||
| Line 13: | Line 14: | ||
*The result cannot be shown now but will be uploaded later. | *The result cannot be shown now but will be uploaded later. | ||
==Plans for tomorrow== | |||
*Miniprep of I7100. | *Miniprep of I7100. | ||
*Digestion test of some other plasmids. | *Digestion test of some other plasmids. | ||
*PCR practice. | *PCR practice. | ||
*Fix the UVP system. | *Fix the UVP system. | ||
=OriT Knock Out= | |||
*By Xu Anting and Liu Ting | |||
==Chromasome Extraction== | |||
#Centrifuged overnight-shaked bacteria and added 700 uL Tris-HCl (pH 8.0) / EDTA (1mM) | |||
#Added Proteiase K (10 mg/mL) 10 uL, then 10% SDS 38 uL to the final volume of 750 uL. | |||
#Hold overnight in 50 centigrade. | |||
Revision as of 22:33, 14 August 2007
Plasmid Construction
Miniprep
- For E0040 and J23066; Note: pSB3K3 fails to grow in the preculture process. Possible reason: this pSB3K3 contains ccdB and the supposed transformants on the pSB3K3 plate are contaminated.
- Use Trans Kit.
- Final volume: 50uL for each plasmids.
Fetch the I7100
- We use I7100, which is inserted in pSB3K3, to substitute the pSB3K3 carrying the ccdB.
Digestion Test
- For pSB1AK3-1, R0040-1 and E0040-2
- EcoR I single digestion.
- Our digestion system contains (totally 20uL): 2uL 10*H, 0.5uL EcoR I, 3uL plasmid, 15uL ddH2O.
- The result cannot be shown now but will be uploaded later.
Plans for tomorrow
- Miniprep of I7100.
- Digestion test of some other plasmids.
- PCR practice.
- Fix the UVP system.
OriT Knock Out
- By Xu Anting and Liu Ting
Chromasome Extraction
- Centrifuged overnight-shaked bacteria and added 700 uL Tris-HCl (pH 8.0) / EDTA (1mM)
- Added Proteiase K (10 mg/mL) 10 uL, then 10% SDS 38 uL to the final volume of 750 uL.
- Hold overnight in 50 centigrade.
