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August/11 August 2009
From 2009.igem.org
1.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
Vector1 1-2M 12 SpeⅠ 1 PstⅠ 1 No.2 Buffer 2 dH2O 4 total 20uL Insert1 2-8M 5 XbaⅠ 1 PstⅠ 1 No.2 2 dH2O 11 total 20 uL Vector2 1-23L 12 EcoRⅠ 1 XbaⅠ 1 No.2 2 dH2O 4 total 20uL Insert2 3-18O 5 EcoⅠ 1 SpeⅠ 1 No.2 2 dH2O 11 total 20uL ↓ 37℃ , 2hr
↓
gel electrophoresis
gel cut
↓
purification by [QIAquick Nucleotide Removal Kit]
↓
ligation
ligation DNA 44 10* buffer 5 ligation 1 total 50uL ↓ 16℃ 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??
