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	<b>Results:</b>Ran samples on a 15% SDS-page. The gel did not show any signs of over-expression.<br>		<b>Results:</b>Ran samples on a 15% SDS-page. The gel did not show any signs of over-expression.<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>

---

Revision as of 02:22, 25 July 2010

Back to Notebook
Back to Lab Work

Contents 1 July 2010 1.1 July 5/2010 1.2 July 5/2010 Evening 1.3 July 6/2010

July 2010

July 5/2010

(In Lab: JV, AV, HB)

Objective: Run a 1% agarose gel of purified PCR samples from June 24/10

Method:

Lane	Sample	Components (µL)
1	1kb Ladder	0.5 Ladder + 2 Dye (6X) + 7.5 Milli-Q H2O
2	1 - pBAD (A4)	1 DNA + 2 Dye (6X) + 7 Milli-Q H2O
3	2 - pBAD (A5)	1 DNA + 2 Dye (6X) + 7 Milli-Q H2O
4	3 - SRBS (A6)	1 DNA + 2 Dye (6X) + 7 Milli-Q H2O
5	4 - SRBS (A7)	1 DNA + 2 Dye (6X) + 7 Milli-Q H2O
6	5 - CFP Complete (A8)	1 DNA + 2 Dye (6X) + 7 Milli-Q H2O
7	6 - SRBS (A10)	1 DNA + 2 Dye (6X) + 7 Milli-Q H2O
8	7 - EYFP (B1)	1 DNA + 2 Dye (6X) + 7 Milli-Q H2O
9	8 - N term tag (B2)	1 DNA + 2 Dye (6X) + 7 Milli-Q H2O
10	9 - pSB NEYFP (B4)	1 DNA + 2 Dye (6X) + 7 Milli-Q H2O
11	10 - pSB NEYFP (B5)	1 DNA + 2 Dye (6X) + 7 Milli-Q H2O
12	11 - CFP (B6)	1 DNA + 2 Dye (6X) + 7 Milli-Q H2O
13	12 - pBAD-TetR (B10)	1 DNA + 2 Dye (6X) + 7 Milli-Q H2O
14	13 - D3	1 DNA + 2 Dye (6X) + 7 Milli-Q H2O
15	14 - C term (D4)	1 DNA + 2 Dye (6X) + 7 Milli-Q H2O
16	15 - C term (D5)	1 DNA + 2 Dye (6X) + 7 Milli-Q H2O
17	16 - pLacI (D6)	1 DNA + 2 Dye (6X) + 7 Milli-Q H2O
18	17 - NEYFP (E2)	1 DNA + 2 Dye (6X) + 7 Milli-Q H2O
19	18 - CEYFP (E6)	1 DNA + 2 Dye (6X) + 7 Milli-Q H2O
20	19 - CEYFP (E7)	1 DNA + 2 Dye (6X) + 7 Milli-Q H2O
1	1kb ladder	0.5 Ladder + 2 Dye (6X) + 7.5 Milli-Q H2O
2	20 - EYFP (E8)	1 DNA + 2 Dye (6X) + 7 Milli-Q H2O
3	21 - EYFP (E9)	1 DNA + 2 Dye (6X) + 7 Milli-Q H2O
4	22 - EYFP (E10)	1 DNA + 2 Dye (6X) + 7 Milli-Q H2O
5	23 - ECFP (F1)	1 DNA + 2 Dye (6X) + 7 Milli-Q H2O
6	24 - ECFP (F2)	1 DNA + 2 Dye (6X) + 7 Milli-Q H2O
7	25 - ECFP (F3)	1 DNA + 2 Dye (6X) + 7 Milli-Q H2O
8	26 - pBAD-TetR (F4)	1 DNA + 2 Dye (6X) + 7 Milli-Q H2O
9	27 - pBAD-TetR (F5)	1 DNA + 2 Dye (6X) + 7 Milli-Q H2O
10	28 - EYFP (G1)	1 DNA + 2 Dye (6X) + 7 Milli-Q H2O
11	29 - pSB CEYFP (G4)	1 DNA + 2 Dye (6X) + 7 Milli-Q H2O
12	30 - pBAD (1) (G6)	1 DNA + 2 Dye (6X) + 7 Milli-Q H2O
13	31 - pBAD (2) (G7)	1 DNA + 2 Dye (6X) + 7 Milli-Q H2O
14	32 - N term tag (G8)	1 DNA + 2 Dye (6X) + 7 Milli-Q H2O
15	33 - lumazine (G9)	1 DNA + 2 Dye (6X) + 7 Milli-Q H2O

Ran gel at 100V for 45 minutes.

July 5/2010 Evening

Objective:To over-express CFP complete in DH5α

Method:

1) Inoculated 6mL culture with ampicillin and glycerol stock A6-May 13,2010 CFP complete 2) Went in the shaker at 6:50pm

July 6/2010

(In lab: JV, AV, HB)
Objective:To continue the over-expression of CFP complete in DH5α

Method:
1) Put both cultures (taken out of shaker at 9:15am) and put them in 500mL of LB w/ Amp.
2) initial OD was 0.071 (600λ)

Issue:

• After checking the the sequencing it was evident that our promotor is always off. It is turned off by the product of the gene TetR. Which is not part of our construct.
  

table>OD (600λ)

Time (hours)
0	0.071
1	0.390
1.5(T0)	0.606
2.5 (T1)	1.250
3.5(T2)	3.04
4.5(T3)	2.75

Results:Ran samples on a 15% SDS-page. The gel did not show any signs of over-expression.