"
August/13 August 2009
From 2009.igem.org
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 Vector1 RBS (1-2M) 10 SpeⅠ 1 PstⅠ 1 No.2 Buffer 2 dH2O 6 total 20uL Insert1 cinR(1-14F) 6 XbaⅠ 1 PstⅠ 1 No.2 2 dH2O 10 total 20 uL K204004 Vector2 RBS (1-2M) 10 EcoRⅠ 1 XbaⅠ 1 No.2 2 dH2O 4 total 20uL Insert2 lasR (2-8M) 10 EcoⅠ 1 SpeⅠ 1 No.2 2 dH2O 6 total 20uL K204005 Vector3 (1-18L) 8 SpeⅠ 1 PstⅠ 1 No.2 Buffer 2 dH2O 8 total 20uL Insert3 (1-23J) 8 XbaⅠ 1 PstⅠ 1 No.2 2 dH2O 8 total 20 uL K204006 Vector4 RBS (1-2M) 10 SpeⅠ 1 PstⅠ 1 No.2 Buffer 2 dH2O 6 total 20uL Insert4 (1-14D) 10 XbaⅠ 1 PstⅠ 1 No.2 2 dH2O 8 total 20 uL ↓ 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.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
2-A 8 XbaⅠ 1 PstⅠ 1 No.2 2 dH2O 8 total 20 uL 2-B 8 XbaⅠ 1 PstⅠ 1 No.2 2 dH2O 8 total 20 uL 2-C 8 XbaⅠ 1 PstⅠ 1 No.2 2 dH2O 8 total 20 uL
1-23L(Vector) 8 XbaⅠ 1 No.2 2 dH2O 9 total 20 uL
↓
37℃ over night
3.2. r apid check
No.3 and No.8 → to iquid medium(5mL + Amp 5uL)
