IGEM:Peking/2007/Count-Conjugation-Notebook/2007-7-17: Difference between revisions
From OpenWetWare
Jump to navigationJump to search
No edit summary |
mNo edit summary |
||
| Line 22: | Line 22: | ||
=OriT Knock Out= | =OriT Knock Out= | ||
*By Xu Anting and Liu Ting | *By Xu Anting and Liu Ting | ||
== | ==Chromosome Extraction== | ||
#Centrifuged overnight-shaked bacteria and added 700 uL Tris-HCl (pH 8.0) / EDTA (1mM) | #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. | #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. | #Hold overnight in 50 centigrade. | ||
Latest revision as of 08:43, 21 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
Chromosome 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.
