Team:Lethbridge/Notebook/Lab Work/June

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==June 2010==
==June 2010==
===June 1/2010===
===June 1/2010===
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JV quantified the amount of DNA in gels run to date using ImageJ software. Results to be posted in [[Team:Lethbridge/Notebook/Working_Plasmids|working plasmids box]].<br><br>
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JV quantified the amount of DNA in gels run to date using ImageJ software. Results to be posted in [[Team:Lethbridge/Notebook/Working_Plasmids|working plasmids box]].<br>
 +
<b>Objective:</b> Transform plasmids into DH5&alpha;<br>
 +
<b>Method:</b> Follow [[Team:Lethbridge/Notebook/Protocols|competent cell transformation]] protocol to transform the following:<br>
 +
From our ligations:<br>
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*pLacI-sRBS-Lumazine-dT<br>
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*pLacI-sRBS-Lumazine-dT<br>
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*mms6 (A6)<br>
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*mms6 (B6)<br>
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*xylE (C4)<br>
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*xylE (B4)<br>
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From the 2010 Parts Distribution:<br>
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*ECFP (Bba_E0020)<br>
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*EYFP (Bba_E0030)<br>
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*BglII Endonuclease (Bba_K112106)<br>
===June 2/2010===
===June 2/2010===

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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 (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

I started ran the reaction for 80 minutes at 37oC.

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.