Team:Lethbridge/Notebook/Lab Work/June

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(In Lab: JV)<br>
(In Lab: JV)<br>
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<b>Objective:</b> Isolate plasmid DNA (BBa_J33204) from DH5&alpha; cells and confirm results.<br>
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<b>Objective:</b> Isolate plasmid DNA of RBS-xylE (BBa_J33204) from DH5&alpha; cells and confirm results.<br>
<b>Method:</b> "Mini-prep" the plasmid DNA using [[Team:Lethbridge/Notebook/Protocols|boiling lysis miniprep]]. Then restrict the DNA once and run on a 1% agarose gel (TAE). <br>
<b>Method:</b> "Mini-prep" the plasmid DNA using [[Team:Lethbridge/Notebook/Protocols|boiling lysis miniprep]]. Then restrict the DNA once and run on a 1% agarose gel (TAE). <br>
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</table> <br>
</table> <br>
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I started ran the reaction for 80 minutes at 37<sup>o</sup>C.<br>
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DNA was restricted for 80 minutes at 37<sup>o</sup>C.<br>
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Analyzed results on a 1% agarose gel. Load order as follows:<br>
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<table><table border ="3">
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<tr><td><b>Lane</b></td><td><b>Sample</b></td><td><b>Volume<br>Sample (&micro;L)</b></td><td>Volume Loading<br>Dye (&micro;L)</b></td></tr>
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<tr><td>1</td><td>Restricted RBS-xylE</td><td>10</td><td>2</td></tr>
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<tr><td>1</td><td>Unestricted RBS-xylE<sup>&dagger;</sup></td><td>1</td><td>2</td></tr>
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<tr><td>1</td><td>1kb Ladder<sup>&dagger;</sup><sup>&dagger;</sup></td><td>2</td><td>2</td></tr></table>
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&dagger; Added 9&micro;L MilliQ H<sub>2</sub>O<br>
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&dagger;&dagger; Added 8&micro;L MilliQ H<sub>2</sub>O<br>
===June 3/2010===
===June 3/2010===
<b>Objective:</b> Ligate pLacI to sRBS-Lum-dT using the three antibiotic assembly method (according to Tom Knight's protocol). Previous ligation had miniscule quantities of DNA in ligation, not surprising it didn't work.<br>
<b>Objective:</b> Ligate pLacI to sRBS-Lum-dT using the three antibiotic assembly method (according to Tom Knight's protocol). Previous ligation had miniscule quantities of DNA in ligation, not surprising it didn't work.<br>

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Contents

June 2010

June 1/2010

JV quantified the amount of DNA in gels run to date using ImageJ software. Results to be posted in working plasmids box.

Objective: Transform plasmids into DH5α
Method: Follow competent cell transformation protocol to transform the following:
From our ligations:

  • pLacI-sRBS-Lumazine-dT
  • pLacI-sRBS-Lumazine-dT
  • mms6 (A6)
  • mms6 (B6)
  • xylE (C4)
  • xylE (B4)

From the 2010 Parts Distribution:

  • ECFP (Bba_E0020)
  • EYFP (Bba_E0030)
  • BglII Endonuclease (Bba_K112106)

June 2/2010

(In Lab: JV)

Objective: Isolate plasmid DNA of RBS-xylE (BBa_J33204) from DH5α cells and confirm results.

Method: "Mini-prep" the plasmid DNA using boiling lysis miniprep. Then restrict the DNA once and run on a 1% agarose gel (TAE).


Restriction Reaction

IngredientVolume(µL)
MilliQ H20 Water15.75
Orange Buffer (10x)2
pDNA (rbs-xylE)2
EcoRI0.25

Unrestricted Control

IngredientVolume(µL)
MilliQ H20 Water16
Orange Buffer (10x)2
pDNA (rbs-xylE)2

DNA was restricted for 80 minutes at 37oC.

Analyzed results on a 1% agarose gel. Load order as follows:

<tr><td>1</td><td>Restricted RBS-xylE</td><td>10</td><td>2</td></tr> <tr><td>1</td><td>Unestricted RBS-xylE†</td><td>1</td><td>2</td></tr> <tr><td>1</td><td>1kb Ladder††</td><td>2</td><td>2</td></tr></table> † Added 9µL MilliQ H2O
†† Added 8µL MilliQ H2O

June 3/2010

Objective: Ligate pLacI to sRBS-Lum-dT using the three antibiotic assembly method (according to Tom Knight's protocol). Previous ligation had miniscule quantities of DNA in ligation, not surprising it didn't work.

Retrieved from "http://2010.igem.org/Team:Lethbridge/Notebook/Lab_Work/June"
LaneSampleVolume
Sample (µL)		Volume Loading
Dye (µL)</b>