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August/11 August 2009
From 2009.igem.org
(Difference between revisions)
| Line 17: | Line 17: | ||
digestion with restriction enzyme | digestion with restriction enzyme | ||
| + | |||
| + | K204001 | ||
Vector1 | Vector1 | ||
1-2M 12 | 1-2M 12 | ||
| Line 34: | Line 36: | ||
| + | K204002 | ||
Vector2 | Vector2 | ||
1-23L 12 | 1-23L 12 | ||
Revision as of 08:10, 14 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 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 K204002 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??
