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.