August/11 August 2009

1.before Min prep Checked the cell cultures transformed and plated out yesterday (8/10) for colony formation and made an approximate count of the number of colonies.

(plate number)-(location on plate) (no. of colonies) 1-23L                    100> 1-15N                     10 2-6P                      10< 1-6I                      10< 1-12H                     10 1-18C                     10< 1-20F                      × inoculate to iquid medium(5mL + Amp 5uL or Kan 25uL)

2.digestion and ligation

digestion with restriction enzyme K204001

K204002

↓ 37°C, 2hr

↓ gel electrophoresis No.1 No.2 ladder gel cut ↓ purification by [QIAquick Nucleotide Removal Kit] ↓ ligation ligation DNA       44 10* buffer 5 ligation  1 total     50uL ↓ 16°C,overnight

3.Transformation to get new plasmid each 4uL(DNA), 1-14F : 2uL 1-14H kan 1-14F kan 2-18F Amp ('8/10competent cell 1-23J Amp 1-12C Amp (super competent cell??

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