IGEM:Paris Bettencourt 2012/Notebooks/suicide group/day by day//2012/08/20

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(Entry title)
Current revision (13:46, 20 August 2012) (view source)
(Entry title)
 
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==Entry title==
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==Transformation==
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* Insert content here...
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*transformed NEB Turbo cells with pSG.008, pSG.017, and pSG.018
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-transformed NEB Turbo cells with pSG.008, pSG.017, and pSG.018
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*prepared liquid cultures for colicin assay
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-prepared liquid cultures for colicin assay
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 +
==Digestion==
 +
We digested pSG.018 (constitutive promoter) with SpeI and PstI; and pSG.013 (ColE2 Toxin) with XbaI and PstI.
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===Protocol===
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 +
{| style="border-spacing:0;"
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| 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;"|
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| 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;"| PSG.018
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| style="border:0.002cm solid #000000;padding:0.097cm;"| PSG.013
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|-
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| style="border-top:none;border-bottom:0.002cm solid #000000;border-left:0.002cm solid #000000;border-right:none;padding:0.097cm;"| Green Buffer
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| style="border-top:none;border-bottom:0.002cm solid #000000;border-left:0.002cm solid #000000;border-right:none;padding:0.097cm;"| 4 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;"| 4 uL
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|-
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| style="border-top:none;border-bottom:0.002cm solid #000000;border-left:0.002cm solid #000000;border-right:none;padding:0.097cm;"| SpeI
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| style="border-top:none;border-bottom:0.002cm solid #000000;border-left:0.002cm solid #000000;border-right:none;padding:0.097cm;"| 2 uL
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| 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;"| -
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|-
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| style="border-top:none;border-bottom:0.002cm solid #000000;border-left:0.002cm solid #000000;border-right:none;padding:0.097cm;"| PstI
 +
| style="border-top:none;border-bottom:0.002cm solid #000000;border-left:0.002cm solid #000000;border-right:none;padding:0.097cm;"| 2 uL
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| 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
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 +
|-
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| style="border-top:none;border-bottom:0.002cm solid #000000;border-left:0.002cm solid #000000;border-right:none;padding:0.097cm;"| XbaI
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| 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;"| 2 uL
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|-
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| style="border-top:none;border-bottom:0.002cm solid #000000;border-left:0.002cm solid #000000;border-right:none;padding:0.097cm;"| Plasmid
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| style="border-top:none;border-bottom:0.002cm solid #000000;border-left:0.002cm solid #000000;border-right:none;padding:0.097cm;"| 6 uL (+/-180 ng/uL)
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| 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;"| 25 uL (+/- 25 ng/uL)
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|-
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| style="border-top:none;border-bottom:0.002cm solid #000000;border-left:0.002cm solid #000000;border-right:none;padding:0.097cm;"| H2O
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| style="border-top:none;border-bottom:0.002cm solid #000000;border-left:0.002cm solid #000000;border-right:none;padding:0.097cm;"| 26 uL
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| 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;"| 7 uL
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 +
|}
 +
 
 +
===Gel===
 +
We gel purified pSG.018 and PCR purified pSG.013 (basically the same just that we ran pSG.018 on the gel)
 +
 
 +
[[Image:200812.png]]
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Transformation

  • transformed NEB Turbo cells with pSG.008, pSG.017, and pSG.018
  • prepared liquid cultures for colicin assay

Digestion

We digested pSG.018 (constitutive promoter) with SpeI and PstI; and pSG.013 (ColE2 Toxin) with XbaI and PstI.

Protocol

PSG.018 PSG.013
Green Buffer 4 uL 4 uL
SpeI 2 uL -
PstI 2 uL 2 uL
XbaI - 2 uL
Plasmid 6 uL (+/-180 ng/uL) 25 uL (+/- 25 ng/uL)
H2O 26 uL 7 uL

Gel

We gel purified pSG.018 and PCR purified pSG.013 (basically the same just that we ran pSG.018 on the gel)

Image:200812.png

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