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August/10 August 2009
From 2009.igem.org
(Difference between revisions)
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| - | 1.Min prep | + | 1.<B>Min prep</b><br> |
| + | Result of Nanodrop concentration check | ||
| - | 1-6O | + | (sample No.) (concentration) |
| - | 1-18L | + | [http://partsregistry.org/wiki/index.php?title=Part:BBa_R0062 1-6O] 35.2 ng/uL |
| - | 1-10K | + | [http://partsregistry.org/wiki/index.php?title=Part:BBa_I14017 1-18L] 41.8 ng/uL |
| + | [http://partsregistry.org/wiki/index.php?title=Part:BBa_R0077 1-10K] 39.1 ng/uL | ||
| - | |||
| - | 3.digestion with | + | 2.<B>make competent cells</b>(with CaCl2) |
| + | |||
| + | |||
| + | |||
| + | 3.<B>digestion and ligation</b><br> | ||
| + | digestion with restriction enzyme | ||
| + | |||
| + | <table border="1" Frame="box"> | ||
| + | <tr><th colspan="2">Vector1</th><th colspan="2">Insert1</th></tr> | ||
| + | <tr><td>[http://partsregistry.org/wiki/index.php?title=Part:BBa_B0015 1-23L]</td><td>12</td><td>[http://partsregistry.org/wiki/index.php?title=Part:BBa_K118003 2-16H]</td><td>6</td></tr> | ||
| + | <tr><td>EcoRI</td><td>1</td><td>EcoRI</td><td>1</td><tr> | ||
| + | <tr><td>SpeI</td><td>1</td><td>XbaI</td><td>1</td><tr> | ||
| + | <tr><td> No.2 Buffer</td><td>2</td><td>No.2</td><td>2</td></tr> | ||
| + | <tr><td>dH<sub>2</sub>O</td><td>4</td><td>dH<sub>2</sub>O</td><td>10</td></tr> | ||
| + | <tr><td>total</td><td>20uL</td><td>total</td><td>20uL</td></tr> | ||
| + | </table> | ||
| + | |||
| + | |||
| + | <table border="1" Frame="box"> | ||
| + | <tr><th colspan="2">Vector2</th><th colspan="2">Insert2</th></tr> | ||
| + | <tr><td>[http://partsregistry.org/wiki/index.php?title=Part:BBa_B0015 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>SpeI</td><td>1</td><td>XbaI</td><td>1</td><tr> | ||
| + | <tr><td> No.2 Buffer</td><td>2</td><td>No.2</td><td>2</td></tr> | ||
| + | <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> | ||
| + | </table> | ||
| + | |||
| + | |||
| + | |||
| + | |||
| + | ↓<br> | ||
| + | [[Image:Osaka090810.jpg]]<br> | ||
| + | vector1 insert1 vector2 insert2 ladder | ||
| + | failure<br> | ||
| + | |||
| + | |||
| + | digestion with restriction enzyme | ||
insert : E+S | insert : E+S | ||
vector : E+x | vector : E+x | ||
| - | |||
| - | 4.Transformation | + | |
| - | 1-23L A | + | |
| - | 1-18C A | + | |
| - | 1-20F K | + | 4.<B>Transformation</b><br> |
| - | 1-12H K | + | to get new plasmid |
| - | 1-6I A | + | [http://partsregistry.org/wiki/index.php?title=Part:BBa_B0015 1-23L] A |
| - | 2-6P A | + | [http://partsregistry.org/wiki/index.php?title=Part:BBa_J23100 1-18C] A |
| - | 1-15N A | + | [http://partsregistry.org/wiki/index.php?title=Part:BBa_I15009 1-20F] K |
| + | [http://partsregistry.org/wiki/index.php?title=Part:BBa_C0070 1-12H] K | ||
| + | [http://partsregistry.org/wiki/index.php?title=Part:BBa_R0040 1-6I] A | ||
| + | [http://partsregistry.org/wiki/index.php?title=Part:BBa_K091101 2-6P] A | ||
| + | [http://partsregistry.org/wiki/index.php?title=Part:BBa_I719005 1-15N] A | ||
used '8/10 competent cell | used '8/10 competent cell | ||
| + | |||
| + | [https://2009.igem.org/Team:Osaka/NOTES back to NOTES] | ||
Latest revision as of 07:43, 12 October 2009
1.Min prep
Result of Nanodrop concentration check
(sample No.) (concentration) [http://partsregistry.org/wiki/index.php?title=Part:BBa_R0062 1-6O] 35.2 ng/uL [http://partsregistry.org/wiki/index.php?title=Part:BBa_I14017 1-18L] 41.8 ng/uL [http://partsregistry.org/wiki/index.php?title=Part:BBa_R0077 1-10K] 39.1 ng/uL
2.make competent cells(with CaCl2)
3.digestion and ligation
digestion with restriction enzyme
| Vector1 | Insert1 | ||
|---|---|---|---|
| [http://partsregistry.org/wiki/index.php?title=Part:BBa_B0015 1-23L] | 12 | [http://partsregistry.org/wiki/index.php?title=Part:BBa_K118003 2-16H] | 6 |
| EcoRI | 1 | EcoRI | 1 |
| SpeI | 1 | XbaI | 1 |
| No.2 Buffer | 2 | No.2 | 2 |
| dH2O | 4 | dH2O | 10 |
| total | 20uL | total | 20uL |
| Vector2 | Insert2 | ||
|---|---|---|---|
| [http://partsregistry.org/wiki/index.php?title=Part:BBa_B0015 1-23L] | 12 | 3-18O | 5 |
| EcoRI | 1 | EcoRI | 1 |
| SpeI | 1 | XbaI | 1 |
| No.2 Buffer | 2 | No.2 | 2 |
| dH2O | 4 | dH2O | 11 |
| total | 20uL | total | 20uL |
↓
vector1 insert1 vector2 insert2 ladder
failure
digestion with restriction enzyme insert : E+S vector : E+x
4.Transformation
to get new plasmid
[http://partsregistry.org/wiki/index.php?title=Part:BBa_B0015 1-23L] A [http://partsregistry.org/wiki/index.php?title=Part:BBa_J23100 1-18C] A [http://partsregistry.org/wiki/index.php?title=Part:BBa_I15009 1-20F] K [http://partsregistry.org/wiki/index.php?title=Part:BBa_C0070 1-12H] K [http://partsregistry.org/wiki/index.php?title=Part:BBa_R0040 1-6I] A [http://partsregistry.org/wiki/index.php?title=Part:BBa_K091101 2-6P] A [http://partsregistry.org/wiki/index.php?title=Part:BBa_I719005 1-15N] A used '8/10 competent cell
