August/13 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) 1-2M                      100~    (Amp) 2-24G                     1? 1-12C                     10 1-6I                      10< 2-8I                      10< 2-6E                      10< 1-16G                     10 1-12A                     10< 2-8O                      10< 2-11N                      10    (Kan) 1-18L                      50 inoculate to iquid medium(5mL + Amp 5uL or Kan 25uL) after ligation EpsE + terminator                   3        →  liquid medium RBS  +  LasR(2-8M)                 10     → rapid check

2.digestion and ligation

digestion with restriction enzyme K204003

K204004

K204005

K204006

↓ 37°C, 2hr

↓ gel electrophoresis No. 3, 4 , 5 , 6 , ladder gel cut ↓ purification by [QIAquick Nucleotide Removal Kit] ↓ ligation ligation DNA       44 10* buffer 5 ligation  1 total     50uL ↓ 16°C overnight

3.ligation check

3.1. EpsE + terminater Result of Nanodrop concentration check (sample No.) (concentration) 2-A              21.5 ng/uL 2-B              15.8 ng/uL 2-C              16.7 ng/uL

↓ digestion with restriction enzyme

↓ 37°C over night 3.2. rapid check (RBS  +  LasR(2-8M)) No.3 and No.8 → to iquid medium(5mL + Amp 5uL)

back to NOTES