"
August/11 August 2009
From 2009.igem.org
(Difference between revisions)
| Line 18: | Line 18: | ||
digestion with restriction enzyme | digestion with restriction enzyme | ||
| - | + | '''K204001''' | |
| - | + | <table border="1" Frame="box"> | |
| - | + | <tr><th colspan="2">Vector</th><th colspan="2">Insert</th></tr> | |
| - | + | <tr><td>1-2M</td><td>12</td><td>2-8M</td><td>5</td></tr> | |
| - | + | <tr><td>SpeI</td><td>1</td><td>XbaI</td><td>1</td><tr> | |
| - | + | <tr><td>PstI</td><td>1</td><td>PstI</td><td>1</td><tr> | |
| - | + | <tr><td> No.2 Buffer</td><td>2</td><td>No.2</td><td>2</td></tr> | |
| - | + | <tr><td>dH2O</td><td>4</td><td>dH2O</td><td>11</td></tr> | |
| + | <tr><td>total</td><td>20uL</td><td>total</td><td>20uL</td></tr> | ||
| + | </table> | ||
| + | |||
| + | |||
| - | + | '''K204002''' | |
| - | + | ||
| - | + | <table border="1" Frame="box"> | |
| - | + | <tr><th colspan="2">Vector</th><th colspan="2">Insert</th></tr> | |
| - | + | <tr><td>1-23L</td><td>12</td><td>3-18O</td><td>5</td></tr> | |
| - | + | <tr><td>EcoRI</td><td>1</td><td>EcoRI</td><td>1</td><tr> | |
| - | + | <tr><td>XbaI</td><td>1</td><td>SpeI</td><td>1</td><tr> | |
| - | + | <tr><td> No.2</td><td>2</td><td>No.2</td><td>2</td></tr> | |
| - | + | <tr><td>dH2O</td><td>4</td><td>dH2O</td><td>11</td></tr> | |
| - | + | <tr><td>total</td><td>20uL</td><td>total</td><td>20uL</td></tr> | |
| - | + | </table> | |
| - | + | ||
| - | + | ↓<br> | |
| - | + | 37°C , 2hr<br> | |
| - | + | ||
| - | + | ||
| - | + | ||
| - | + | ||
| - | + | ||
| - | + | ||
| - | + | ||
| - | + | ||
| - | + | ||
| - | + | ||
| - | + | ||
| - | + | ||
| - | + | ||
| - | + | ||
↓<br> | ↓<br> | ||
| Line 69: | Line 59: | ||
total 50uL | total 50uL | ||
↓ | ↓ | ||
| - | + | 16°C,overnight | |
Revision as of 11:29, 16 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
| Vector | Insert | ||
|---|---|---|---|
| 1-2M | 12 | 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 | ||
|---|---|---|---|
| 1-23L | 12 | 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
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??
