IGEM:Paris Bettencourt 2012/Notebooks/suicide group/day by day//2012/09/07: Difference between revisions
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==Biobricks== | |||
We put pSG.008 (pLac+ColE2 immunity) and pSG.014 (ColE2 immunity) inside Biobricks vector (pSB1C3). All of them were cut with XbaI and PstI. | |||
===Protocol=== | |||
{| style="border-spacing:0;" | |||
| style="border-top:0.002cm solid #000000;border-bottom:0.002cm solid #000000;border-left:0.002cm solid #000000;border-right:none;padding:0.097cm;"| | |||
| style="border-top:0.002cm solid #000000;border-bottom:0.002cm solid #000000;border-left:0.002cm solid #000000;border-right:none;padding:0.097cm;"| PSB1C3 (-) | |||
| style="border-top:0.002cm solid #000000;border-bottom:0.002cm solid #000000;border-left:0.002cm solid #000000;border-right:none;padding:0.097cm;"| BB+pSG.008 | |||
| style="border:0.002cm solid #000000;padding:0.097cm;"| BB+pSG.014 | |||
|- | |||
| style="border-top:none;border-bottom:0.002cm solid #000000;border-left:0.002cm solid #000000;border-right:none;padding:0.097cm;"| H2O | |||
| style="border-top:none;border-bottom:0.002cm solid #000000;border-left:0.002cm solid #000000;border-right:none;padding:0.097cm;"| 6.5 uL | |||
| style="border-top:none;border-bottom:0.002cm solid #000000;border-left:0.002cm solid #000000;border-right:none;padding:0.097cm;"| - | |||
| style="border-top:none;border-bottom:0.002cm solid #000000;border-left:0.002cm solid #000000;border-right:0.002cm solid #000000;padding:0.097cm;"| - | |||
|- | |||
| style="border-top:none;border-bottom:0.002cm solid #000000;border-left:0.002cm solid #000000;border-right:none;padding:0.097cm;"| Insert | |||
| style="border-top:none;border-bottom:0.002cm solid #000000;border-left:0.002cm solid #000000;border-right:none;padding:0.097cm;"| 2 uL | |||
| style="border-top:none;border-bottom:0.002cm solid #000000;border-left:0.002cm solid #000000;border-right:none;padding:0.097cm;"| 2 uL | |||
| style="border-top:none;border-bottom:0.002cm solid #000000;border-left:0.002cm solid #000000;border-right:0.002cm solid #000000;padding:0.097cm;"| 2 uL | |||
|- | |||
| style="border-top:none;border-bottom:0.002cm solid #000000;border-left:0.002cm solid #000000;border-right:none;padding:0.097cm;"| Vector | |||
| style="border-top:none;border-bottom:0.002cm solid #000000;border-left:0.002cm solid #000000;border-right:none;padding:0.097cm;"| - | |||
| style="border-top:none;border-bottom:0.002cm solid #000000;border-left:0.002cm solid #000000;border-right:none;padding:0.097cm;"| 6.5 uL | |||
| style="border-top:none;border-bottom:0.002cm solid #000000;border-left:0.002cm solid #000000;border-right:0.002cm solid #000000;padding:0.097cm;"| 6.5 uL | |||
|- | |||
| style="border-top:none;border-bottom:0.002cm solid #000000;border-left:0.002cm solid #000000;border-right:none;padding:0.097cm;"| Ligase buffer 10x | |||
| style="border-top:none;border-bottom:0.002cm solid #000000;border-left:0.002cm solid #000000;border-right:none;padding:0.097cm;"| 1.0 uL | |||
| style="border-top:none;border-bottom:0.002cm solid #000000;border-left:0.002cm solid #000000;border-right:none;padding:0.097cm;"| 1.0 uL | |||
| style="border-top:none;border-bottom:0.002cm solid #000000;border-left:0.002cm solid #000000;border-right:0.002cm solid #000000;padding:0.097cm;"| 1.0 uL | |||
|- | |||
| style="border-top:none;border-bottom:0.002cm solid #000000;border-left:0.002cm solid #000000;border-right:none;padding:0.097cm;"| T4 ligase enzyme | |||
| style="border-top:none;border-bottom:0.002cm solid #000000;border-left:0.002cm solid #000000;border-right:none;padding:0.097cm;"| 0.5 uL | |||
| style="border-top:none;border-bottom:0.002cm solid #000000;border-left:0.002cm solid #000000;border-right:none;padding:0.097cm;"| 0.5 uL | |||
| style="border-top:none;border-bottom:0.002cm solid #000000;border-left:0.002cm solid #000000;border-right:0.002cm solid #000000;padding:0.097cm;"| 0.5 uL | |||
|- | |||
| style="border-top:none;border-bottom:0.002cm solid #000000;border-left:0.002cm solid #000000;border-right:none;padding:0.097cm;"| Total | |||
| style="border-top:none;border-bottom:0.002cm solid #000000;border-left:0.002cm solid #000000;border-right:none;padding:0.097cm;"| 10 uL | |||
| style="border-top:none;border-bottom:0.002cm solid #000000;border-left:0.002cm solid #000000;border-right:none;padding:0.097cm;"| 10 uL | |||
| style="border-top:none;border-bottom:0.002cm solid #000000;border-left:0.002cm solid #000000;border-right:0.002cm solid #000000;padding:0.097cm;"| 10 uL | |||
|} | |||
===Transformation=== | |||
We transformed 5 uL of each ligation product with heat shock transformation; +pSG.001 as a positive control | |||
===Gel=== | |||
We ran the rest of the ligation product + 3 uL of green buffer; in a gel | |||
[[Image:070912.png]] | |||
__NOTOC__ | __NOTOC__ | ||
[[category:OWWLabNotebookV1]] | [[category:OWWLabNotebookV1]] | ||
Revision as of 10:23, 7 September 2012
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Biobricks
We put pSG.008 (pLac+ColE2 immunity) and pSG.014 (ColE2 immunity) inside Biobricks vector (pSB1C3). All of them were cut with XbaI and PstI.
Protocol
| PSB1C3 (-) | BB+pSG.008 | BB+pSG.014 | |
| H2O | 6.5 uL | - | - |
| Insert | 2 uL | 2 uL | 2 uL |
| Vector | - | 6.5 uL | 6.5 uL |
| Ligase buffer 10x | 1.0 uL | 1.0 uL | 1.0 uL |
| T4 ligase enzyme | 0.5 uL | 0.5 uL | 0.5 uL |
| Total | 10 uL | 10 uL | 10 uL |
Transformation
We transformed 5 uL of each ligation product with heat shock transformation; +pSG.001 as a positive control
Gel
We ran the rest of the ligation product + 3 uL of green buffer; in a gel
