Team:Lethbridge/Notebook/Lab Work/June

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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

Ingredient	Volume(µL)
MilliQ H₂0 Water	15.75
Orange Buffer (10x)	2
pDNA (rbs-xylE)	2
EcoRI	0.25

Unrestricted Control

Ingredient	Volume(µL)
MilliQ H₂0 Water	16
Orange Buffer (10x)	2
pDNA (rbs-xylE)	2

DNA was restricted for 80 minutes at 37^oC.

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.

Lane

Sample

Volume
Sample (µL)

Volume Loading
Dye (µL)</b>

@@ Line 60: / Line 60: @@
 (In Lab: JV)<br>
-<b>Objective:</b> Isolate plasmid DNA (BBa_J33204) from DH5&alpha; cells and confirm results.<br>
+<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>
@@ Line 82: / Line 82: @@
 </table> <br>
-I started ran the reaction for 80 minutes at 37<sup>o</sup>C.<br>
+DNA was restricted for 80 minutes at 37<sup>o</sup>C.<br>
+Analyzed results on a 1% agarose gel. Load order as follows:<br>
+<table><table border ="3">
+<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>
+<tr><td>1</td><td>Restricted RBS-xylE</td><td>10</td><td>2</td></tr>
+<tr><td>1</td><td>Unestricted RBS-xylE<sup>&dagger;</sup></td><td>1</td><td>2</td></tr>
+<tr><td>1</td><td>1kb Ladder<sup>&dagger;</sup><sup>&dagger;</sup></td><td>2</td><td>2</td></tr></table>
+&dagger; Added 9&micro;L MilliQ H<sub>2</sub>O<br>
+&dagger;&dagger; Added 8&micro;L MilliQ H<sub>2</sub>O<br>
 ===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>