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August/11 August 2009
From 2009.igem.org
(Difference between revisions)
| (3 intermediate revisions not shown) | |||
| Line 3: | Line 3: | ||
(plate number)-(location on plate) (no. of colonies) | (plate number)-(location on plate) (no. of colonies) | ||
| - | 1-23L 100> | + | [http://partsregistry.org/wiki/index.php?title=Part:BBa_B0015 1-23L] 100> |
| - | 1-15N 10 | + | [http://partsregistry.org/wiki/index.php?title=Part:BBa_I719005 1-15N] 10 |
| - | 2-6P 10< | + | [http://partsregistry.org/wiki/index.php?title=Part:BBa_K091101 2-6P] 10< |
| - | 1-6I 10< | + | [http://partsregistry.org/wiki/index.php?title=Part:BBa_R0040 1-6I] 10< |
| - | 1-12H 10 | + | [http://partsregistry.org/wiki/index.php?title=Part:BBa_C0070 1-12H] 10 |
| - | 1-18C 10< | + | [http://partsregistry.org/wiki/index.php?title=Part:BBa_J23100 1-18C] 10< |
| - | 1-20F × | + | [http://partsregistry.org/wiki/index.php?title=Part:BBa_I15009 1-20F] × |
inoculate to iquid medium(5mL + Amp 5uL or Kan 25uL) | inoculate to iquid medium(5mL + Amp 5uL or Kan 25uL) | ||
| Line 21: | Line 21: | ||
<table border="1" Frame="box"> | <table border="1" Frame="box"> | ||
<tr><th colspan="2">Vector</th><th colspan="2">Insert</th></tr> | <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>[http://partsregistry.org/wiki/index.php?title=Part:BBa_B0034 RBS] (1-2M)</td><td>12</td><td>[http://partsregistry.org/wiki/index.php?title=Part:BBa_C0179 LasR] (2-8M)</td><td>5</td></tr> |
<tr><td>SpeI</td><td>1</td><td>XbaI</td><td>1</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>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> No.2 Buffer</td><td>2</td><td>No.2</td><td>2</td></tr> | ||
| - | <tr><td> | + | <tr><td>dH<sub>2</sub>O</td><td>4</td><td>dH<sub>2</sub>O</td><td>11</td></tr> |
<tr><td>total</td><td>20uL</td><td>total</td><td>20uL</td></tr> | <tr><td>total</td><td>20uL</td><td>total</td><td>20uL</td></tr> | ||
</table> | </table> | ||
| Line 35: | Line 35: | ||
<table border="1" Frame="box"> | <table border="1" Frame="box"> | ||
<tr><th colspan="2">Vector</th><th colspan="2">Insert</th></tr> | <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>[http://partsregistry.org/wiki/index.php?title=Part:BBa_B0015 terminator] (1-23L)</td><td>12</td><td>[http://partsregistry.org/Part:BBa_K143032 EpsE] (3-18O)</td><td>5</td></tr> |
<tr><td>EcoRI</td><td>1</td><td>EcoRI</td><td>1</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>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> No.2</td><td>2</td><td>No.2</td><td>2</td></tr> | ||
| - | <tr><td> | + | <tr><td>dH<sub>2</sub>O</td><td>4</td><td>dH<sub>2</sub>O</td><td>11</td></tr> |
<tr><td>total</td><td>20uL</td><td>total</td><td>20uL</td></tr> | <tr><td>total</td><td>20uL</td><td>total</td><td>20uL</td></tr> | ||
</table> | </table> | ||
| Line 48: | Line 48: | ||
↓<br> | ↓<br> | ||
gel electrophoresis<br> | gel electrophoresis<br> | ||
| + | [[Image:Osaka090811.jpg]]<br> | ||
| + | No.1 No.2 ladder | ||
gel cut<br> | gel cut<br> | ||
↓<br> | ↓<br> | ||
| Line 65: | Line 67: | ||
to get new plasmid | to get new plasmid | ||
each 4uL(DNA) , 1-14F : 2uL | each 4uL(DNA) , 1-14F : 2uL | ||
| - | 1-14H kan | + | [http://partsregistry.org/wiki/index.php?title=Part:BBa_C0078 1-14H] kan |
| - | 1-14F kan | + | [http://partsregistry.org/wiki/index.php?title=Part:BBa_C0077 1-14F] kan |
| - | 2-18F Amp ('8/10competent cell | + | [http://partsregistry.org/wiki/index.php?title=Part:BBa_K118013 2-18F] Amp ('8/10competent cell |
| - | 1-23J Amp | + | [http://partsregistry.org/wiki/index.php?title=Part:BBa_I1466 1-23J] Amp |
| - | 1-12C Amp (super competent cell?? | + | [http://partsregistry.org/wiki/index.php?title=Part:BBa_R0071 1-12C] Amp (super competent cell?? |
| + | |||
| + | |||
| + | [https://2009.igem.org/Team:Osaka/NOTES back to NOTES] | ||
Latest revision as of 07:46, 12 October 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)
[http://partsregistry.org/wiki/index.php?title=Part:BBa_B0015 1-23L] 100>
[http://partsregistry.org/wiki/index.php?title=Part:BBa_I719005 1-15N] 10
[http://partsregistry.org/wiki/index.php?title=Part:BBa_K091101 2-6P] 10<
[http://partsregistry.org/wiki/index.php?title=Part:BBa_R0040 1-6I] 10<
[http://partsregistry.org/wiki/index.php?title=Part:BBa_C0070 1-12H] 10
[http://partsregistry.org/wiki/index.php?title=Part:BBa_J23100 1-18C] 10<
[http://partsregistry.org/wiki/index.php?title=Part:BBa_I15009 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
[http://partsregistry.org/wiki/index.php?title=Part:BBa_C0078 1-14H] kan
[http://partsregistry.org/wiki/index.php?title=Part:BBa_C0077 1-14F] kan
[http://partsregistry.org/wiki/index.php?title=Part:BBa_K118013 2-18F] Amp ('8/10competent cell
[http://partsregistry.org/wiki/index.php?title=Part:BBa_I1466 1-23J] Amp
[http://partsregistry.org/wiki/index.php?title=Part:BBa_R0071 1-12C] Amp (super competent cell??
