IGEM:Peking/2007/Count-Conjugation-Notebook/2007-7-17

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

1. Centrifuged overnight-shaked bacteria and added 700 uL Tris-HCl (pH 8.0) / EDTA (1mM)
2. Added Proteiase K (10 mg/mL) 10 uL, then 10% SDS 38 uL to the final volume of 750 uL.
3. Hold overnight in 50 centigrade.