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July 2010

June 5/2010

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:

Lane	Sample	Volume Sample (µL)	Volume Loading Dye (µL)
1	Restricted RBS-xylE	10	2
1	Unestricted RBS-xylE^†	1	2
1	1kb Ladder^†^†	2	2

† Added 9µL MilliQ H₂O
†† Added 8µL MilliQ H₂O
Ran gel at 100V from 2 hours.
Results:

Conclusions: Plasmid DNA prep and restriction was successful.

Objective: Ligate rbs-xylE (Bba_J33204) to our double terminator, and insert it into the pSB1T3 plasmid backbone.
Method:

Restrictions
- Restrict rbs-xylE wit EcoRI and SpeI (Red Buffer)
- Restrict the double terminator with XbaI and PstI (Tango Buffer)
- Restrict pSB1T3 with EcoRI and PstI (Red Buffer)
Set up reactions as follows:

Component Volume (µL)

MilliQ H₂O 15.5

Buffer 2

pDNA 2

Enzyme 0.25 + 0.25

Set up control reaction as follows:
- MilliQ H₂O - 16µL
- Buffer - 2µL
- pDNA - 2µL
Incubated reactions for 65 minutes at 37^oC
Killed enzymes by incubating reactions for 10 minutes at 65^oC

Ligation
Reaction set up as follows:

T4 DNA ligase - 0.25µL
rbs-xylE - 5µL
dT - 3µL
pSB1T3 - 8µL
10x Ligation Buffer - 2µL
MilliQ H₂O - 1.75µL

Incubated reactions overnight at room temperature (total of 19.5 hours)
Killed enzymes by incubating reactions for 10 minutes at 80^oC</ul>

June 2/2010 - Evening

Objective: Set up new ligations of pLacI and sRBS-Lum-dT according to Tom Knight's protocol. Previous ligation had very little DNA.
Relevant Information:

Want a final mass of 25ng of each pDNA in the ligation mix.
Final concentration of pDNA in restriction digest should be 25-50ng/µL.
Tom Knight's restriction reaction is 50µL, therefore there should be 1000ng pDNA in each restriction digest.
Identified the following plasmids in our working plasmids box:

Common Name	Location	Concentration (ng/µL)	Volume/rxn (µL)
pLacI Maxiprep	A9	990	~1
pLacI (B1)	A6	440	~2
sRBS-Lum-dT (2)	A1	965	~1
sRBS-Lum-dT (1)	A2	1145	~1
sRBS-Lum-dT Maxiprep	B8	4780	~.2
sRBS-Lum-dT	B7	4375	~.25
sRBS-Lum-dT (1)	G2	335	~3
sRBS-Lum-dT (2)	G3	965	~2

Make a 1:10 dilution of sRBS-Lum-dt maxiprep (D8) and sRBS-Lum-dT (B7). 0.5µL pDNA in 4.5µL water.
Cut pLacI with EcoRI and SpeI
Cut sRBS-Lum-dT with XbaI and PstI
Cut pSB1T3 with EcoRI and PstI
Will have total of 12 ligation reactions, want 12x2µL of pSB1T3 to add to each, therefore want 25µL of pSB1T3.

Method:
Restriction

Name	[pDNA] (ng/µL)	Volume pDNA (µL)	Volume Water (µL)	Volume Buffer (µL)	Enzymes	Total Volume
sRBS-Lum-dT (A1)	965	1	43.5	5	0.25µL XbaI 0.25µL PstI	50
sRBS-Lum-dT (A2)	1145	1	43.5	5	0.25µL XbaI 0.25µL PstI	50
pLacI Maxiprep (A1)	990	1	43.5	5	0.25µL EcoRI 0.25µL SpeI	50
sRBS-Lum-dT Maxiprep(B8)	4780	2 (of 1:10 dilution)	42.5	5	0.25µL XbaI 0.25µL PstI	50
sRBS-Lum-dT (B7)	4375	2.5 (of 1:10 dilution)	42	5	0.25µL XbaI 0.25µL PstI	50
pLacI (D6)	440	2	42.5	5	0.25µL EcoRI 0.25µL SpeI	50
sRBS-Lum-dT (G2)	335	3	41.5	5	0.25µL XbaI 0.25µL PstI	50
sRBS-Lum-dT (G3)	540	2	42.5	5	0.25µL XbaI 0.25µL PstI	50
pSB1T3	25	12.5	7	5	0.25µL EcoRI 0.25µL PstI	50

Incubate for 30 minutes at 37^oC (Start- 12:10pm; End- 12:40pm)
Heat kill enzymes at 80^oC for 20 minutes

Ligation:
In a 10µL final volume, add:

2µL of sRBS-Lum-dT component
2µL of pLacI component
2µL of pSB1T3 component
1µL of T4 Buffer
0.25µL of T4 DNA Ligase
2.75µL of MilliQ H₂O

Incubate for 30 minutes at room temperature to ligate

Incubate for 20 minutes at 80^oC to heat kill

@@ Line 49: / Line 49: @@
 Back to [[Team:Lethbridge/Notebook|Notebook]]<br>
 Back to [[Team:Lethbridge/Notebook/Lab_Work|Lab Work]]
+=July 2010=
+==June 5/2010==
+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>
+*pLacI-sRBS-Lumazine-dT<br>
+*pLacI-sRBS-Lumazine-dT<br>
+*mms6 (A6)<br>
+*mms6 (B6)<br>
+*xylE (C4)<br>
+*xylE (B4)<br>
+From the 2010 Parts Distribution:<br>
+*ECFP (Bba_E0020)<br>
+*EYFP (Bba_E0030)<br>
+*BglII Endonuclease (Bba_K112106)<br>
+==June 2/2010==
+(In Lab: JV)<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>
+Restriction Reaction
+<table><table border ="3">
+<tr><td><b>Ingredient</b></td><td>Volume(&micro;L)</td></tr>
+<tr><td>MilliQ H<sub>2</sub>0 Water</td><td>15.75</td></tr>
+<tr><td>Orange Buffer (10x)</td><td>2</td></tr>
+<tr><td>pDNA (rbs-xylE)</td><td>2</td></tr>
+<tr><td>EcoRI</td><td>0.25</td></tr>
+</table>
+Unrestricted Control
+<table><table border ="3">
+<tr><td><b>Ingredient</b></td><td>Volume(&micro;L)</td></tr>
+<tr><td>MilliQ H<sub>2</sub>0 Water</td><td>16</td></tr>
+<tr><td>Orange Buffer (10x)</td><td>2</td></tr>
+<tr><td>pDNA (rbs-xylE)</td><td>2</td></tr>
+</table> <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><b>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>
+Ran gel at 100V from 2 hours.<br>
+<b>Results:</b> <br>
+[[image:100602 JV rbs-xylE.JPG|75px|none]]<br>
+<b>Conclusions:</b> Plasmid DNA prep and restriction was successful.<br><br>
+<b>Objective:</b> Ligate rbs-xylE (Bba_J33204) to our double terminator, and insert it into the pSB1T3 plasmid backbone.<br>
+<b>Method:</b><br>
+<ul>
+<li><u>Restrictions</u><br>
+*Restrict rbs-xylE wit EcoRI and SpeI (Red Buffer)<br>
+*Restrict the double terminator with XbaI and PstI (Tango Buffer)<br>
+*Restrict pSB1T3 with EcoRI and PstI (Red Buffer)<br>
+Set up reactions as follows:<br>
+<table><table border ="3">
+<tr><td><b>Component</b></td><td><b>Volume (&micro;L)</b></td></tr>
+<tr><td>MilliQ H<sub>2</sub>O</td><td>15.5</td></tr>
+<tr><td>Buffer</td><td>2</td></tr>
+<tr><td>pDNA</td><td>2</td></tr>
+<tr><td>Enzyme</td><td>0.25 + 0.25</td></tr></table>
+Set up control reaction as follows:
+*MilliQ H<sub>2</sub>O - 16&micro;L<br>
+*Buffer - 2&micro;L<br>
+*pDNA - 2&micro;L<br>
+Incubated reactions for 65 minutes at 37<sup>o</sup>C<br>
+Killed enzymes by incubating reactions for 10 minutes at 65<sup>o</sup>C<br>
+<li><u>Ligation</u><br>
+Reaction set up as follows:
+*T4 DNA ligase - 0.25&micro;L<br>
+*rbs-xylE - 5&micro;L<br>
+*dT - 3&micro;L<br>
+*pSB1T3 - 8&micro;L<br>
+*10x Ligation Buffer - 2&micro;L<br>
+*MilliQ H<sub>2</sub>O - 1.75&micro;L<br>
+Incubated reactions overnight at room temperature (total of 19.5 hours)<br>
+Killed enzymes by incubating reactions for 10 minutes at 80<sup>o</sup>C</ul>
+==June 2/2010 - Evening==
+<b>Objective:</b> Set up new ligations of pLacI and sRBS-Lum-dT according to Tom Knight's protocol. Previous ligation had very little DNA.<br>
+<b>Relevant Information:</b><br>
+*Want a final mass of 25ng of each pDNA in the ligation mix.
+*Final concentration of pDNA in restriction digest should be 25-50ng/&micro;L.
+*Tom Knight's restriction reaction is 50&micro;L, therefore there should be 1000ng pDNA in each restriction digest.
+*Identified the following plasmids in our [[Team:Lethbridge/Notebook/Working_Plasmids|working plasmids box]]:
+<table><table border ="3">
+<tr><td><b>Common Name</b></td><td><b>Location</b></td><td><b>Concentration (ng/&micro;L)</b></td><td><b>Volume/rxn (&micro;L)</b></td></tr>
+<tr><td>pLacI Maxiprep</td><td>A9</td><td>990</td><td>~1</td></tr>
+<tr><td>pLacI (B1)</td><td>A6</td><td>440</td><td>~2</td></tr>
+<tr><td>sRBS-Lum-dT (2)</td><td>A1</td><td>965</td><td>~1</td></tr>
+<tr><td>sRBS-Lum-dT (1)</td><td>A2</td><td>1145</td><td>~1</td></tr>
+<tr><td>sRBS-Lum-dT Maxiprep</td><td>B8</td><td>4780</td><td>~.2</td></tr>
+<tr><td>sRBS-Lum-dT</td><td>B7</td><td>4375</td><td>~.25</td></tr>
+<tr><td>sRBS-Lum-dT (1)</td><td>G2</td><td>335</td><td>~3</td></tr>
+<tr><td>sRBS-Lum-dT (2)</td><td>G3</td><td>965</td><td>~2</td></tr></table>
+*Make a 1:10 dilution  of sRBS-Lum-dt maxiprep (D8) and sRBS-Lum-dT (B7). 0.5&micro;L pDNA in 4.5&micro;L water.
+*Cut pLacI with EcoRI and SpeI
+*Cut sRBS-Lum-dT with XbaI and PstI
+*Cut pSB1T3 with EcoRI and PstI
+*Will have total of 12 ligation reactions, want 12x2&micro;L of pSB1T3 to add to each, therefore want 25&micro;L of pSB1T3.
+<b>Method:</b><br>
+<u>Restriction</u><br>
+<table><table border ="3">
+<tr><td><b>Name</b></td><td><b>[pDNA] (ng/&micro;L)</b></td><td><b>Volume<br>pDNA (&micro;L)</b></td><td><b>Volume<br>Water (&micro;L)</b></td><td><b>Volume<br>Buffer (&micro;L)</b></td><td><b>Enzymes</b></td><td><b>Total Volume</b></td></tr>
+<tr><td>sRBS-Lum-dT (A1)</td><td>965</td><td>1</td><td>43.5</td><td>5</td><td>0.25&micro;L XbaI<br>0.25&micro;L PstI</td><td>50</td></tr>
+<tr><td>sRBS-Lum-dT (A2)</td><td>1145</td><td>1</td><td>43.5</td><td>5</td><td>0.25&micro;L XbaI<br>0.25&micro;L PstI</td><td>50</td></tr>
+<tr><td>pLacI Maxiprep (A1)</td><td>990</td><td>1</td><td>43.5</td><td>5</td><td>0.25&micro;L EcoRI<br>0.25&micro;L SpeI</td><td>50</td></tr>
+<tr><td>sRBS-Lum-dT Maxiprep(B8)</td><td>4780</td><td>2 (of 1:10 dilution)</td><td>42.5</td><td>5</td><td>0.25&micro;L XbaI<br>0.25&micro;L PstI</td><td>50</td></tr>
+<tr><td>sRBS-Lum-dT (B7)</td><td>4375</td><td>2.5 (of 1:10 dilution)</td><td>42</td><td>5</td><td>0.25&micro;L XbaI<br>0.25&micro;L PstI</td><td>50</td></tr>
+<tr><td>pLacI (D6)</td><td>440</td><td>2</td><td>42.5</td><td>5</td><td>0.25&micro;L EcoRI<br>0.25&micro;L SpeI</td><td>50</td></tr>
+<tr><td>sRBS-Lum-dT (G2)</td><td>335</td><td>3</td><td>41.5</td><td>5</td><td>0.25&micro;L XbaI<br>0.25&micro;L PstI</td><td>50</td></tr>
+<tr><td>sRBS-Lum-dT (G3)</td><td>540</td><td>2</td><td>42.5</td><td>5</td><td>0.25&micro;L XbaI<br>0.25&micro;L PstI</td><td>50</td></tr>
+<tr><td>pSB1T3</td><td>25</td><td>12.5</td><td>7</td><td>5</td><td>0.25&micro;L EcoRI<br>0.25&micro;L PstI</td><td>50</td></tr></table>
+Incubate for 30 minutes at 37<sup>o</sup>C (Start- 12:10pm; End- 12:40pm)<br>
+Heat kill enzymes at 80<sup>o</sup>C for 20 minutes<br>
+<u>Ligation:</u><br>
+In a 10&micro;L final volume, add:
+*2&micro;L of sRBS-Lum-dT component
+*2&micro;L of pLacI component
+*2&micro;L of pSB1T3 component
+*1&micro;L of T4 Buffer
+*0.25&micro;L of T4 DNA Ligase
+*2.75&micro;L of MilliQ H<sub>2</sub>O
+Incubate for 30 minutes at room temperature to ligate<br>
+Incubate for 20 minutes at 80<sup>o</sup>C to heat kill<br>

Team:Lethbridge/Notebook/Lab Work/July