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August/11 August 2009
From 2009.igem.org
(Difference between revisions)
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gel electrophoresis<br> | gel electrophoresis<br> | ||
| + | [[Image:Osaka090811.jpg]]<br> | ||
| + | No.1 No.2 ladder | ||
gel cut<br> | gel cut<br> | ||
↓<br> | ↓<br> | ||
Revision as of 08:56, 26 September 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
| Vector | Insert | ||
|---|---|---|---|
| [http://partsregistry.org/wiki/index.php?title=Part:BBa_B0034 RBS] (1-2M) | 12 | [http://partsregistry.org/wiki/index.php?title=Part:BBa_C0179 LasR] (2-8M) | 5 |
| SpeI | 1 | XbaI | 1 |
| PstI | 1 | PstI | 1 |
| No.2 Buffer | 2 | No.2 | 2 |
| dH2O | 4 | dH2O | 11 |
| total | 20uL | total | 20uL |
K204002
| Vector | Insert | ||
|---|---|---|---|
| [http://partsregistry.org/wiki/index.php?title=Part:BBa_B0015 terminator] (1-23L) | 12 | [http://partsregistry.org/Part:BBa_K143032 EpsE] (3-18O) | 5 |
| EcoRI | 1 | EcoRI | 1 |
| XbaI | 1 | SpeI | 1 |
| No.2 | 2 | No.2 | 2 |
| dH2O | 4 | dH2O | 11 |
| total | 20uL | total | 20uL |
↓
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??
