Team:Lethbridge/Notebook/Lab Work/July

From 2010.igem.org

(Difference between revisions)
(July 5/2010)
(July 5/2010)
Line 95: Line 95:
<tr><td>15</td><td>33 - lumazine (G9)</td><td>1 DNA + 2 Dye (6X) + 7 Milli-Q H<sub>2</sub>O</td></tr>
<tr><td>15</td><td>33 - lumazine (G9)</td><td>1 DNA + 2 Dye (6X) + 7 Milli-Q H<sub>2</sub>O</td></tr>
</table><br>
</table><br>
 +
 +
Ran gel at 100V for 45 minutes.<br>
 +
 +
==July 5/2010 Evening==
 +
 +
<b>Objective:</b>To over-express CFP complete in DH5&alpha;<br>
 +
 +
<b>Method:</b><br>
 +
 +
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)<br>
 +
<b>Objective:</b>To continue the over-expression of CFP complete in DH5&alpha;<br>
 +
 +
<b>Method:</b><br>
 +
1) Put both cultures (taken out of shaker at 9:15am) and put them in 500mL of LB w/ Amp.<br>
 +
2) initial OD was 0.071 (600&lambda;)<br>
 +
 +
Issue:<br>
 +
*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. <br>
 +
 +
table><table border ="3">
 +
<tr><td><b>Time (hours)</b></td>OD (600&lambda;)</tr>
 +
<tr><td>0</td><td>0.071</td></tr>
 +
<tr><td>1</td><td>0.390</td></tr>
 +
<tr><td>1.5(T<sub>0</sub>)</td><td>0.606</td></tr>
 +
<tr><td>2.5 (T<sub>1</sub>)</td><td>1.250</td></tr>
 +
<tr><td>3.5(T<sub>2</sub>)</td><td>3.04</td></tr>
 +
<tr><td>4.5(T<sub>3</sub>)</td><td>2.75</td></tr>
 +
</table><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==
==June 2/2010==

Revision as of 23:04, 19 July 2010

UofLteamlogo.jpg UofLLabWork.JPG UofLteamlogo.jpg

[http://2010.igem.org/wiki/index.php?title=Team:Lethbridge&Home UofLHome.jpg]

[http://2010.igem.org/Team:Lethbridge/Team UofLTeam.jpg]

[http://2010.igem.org/Team:Lethbridge/Project UofLProjectbutton.jpg]

[http://2010.igem.org/Team:Lethbridge/Notebook UofLNotebookbutton.jpg]

[http://2010.igem.org/Team:Lethbridge/Parts UofLPartsSubmittedToTheRegistrybutton.jpg]

[http://2010.igem.org/Team:Lethbridge/Modeling UofLModelingbutton.jpg]

[http://2010.igem.org/Team:Lethbridge/Ethics UofLEthicsbutton.jpg]

[http://2010.igem.org/Team:Lethbridge/Safety UofLSafetybutton.jpg]

Back to Notebook
Back to Lab Work

Contents

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:

LaneSampleComponents (µL)
11kb Ladder0.5 Ladder + 2 Dye (6X) + 7.5 Milli-Q H2O
21 - pBAD (A4)1 DNA + 2 Dye (6X) + 7 Milli-Q H2O
32 - pBAD (A5)1 DNA + 2 Dye (6X) + 7 Milli-Q H2O
43 - SRBS (A6)1 DNA + 2 Dye (6X) + 7 Milli-Q H2O
54 - SRBS (A7)1 DNA + 2 Dye (6X) + 7 Milli-Q H2O
65 - CFP Complete (A8)1 DNA + 2 Dye (6X) + 7 Milli-Q H2O
76 - SRBS (A10)1 DNA + 2 Dye (6X) + 7 Milli-Q H2O
87 - EYFP (B1)1 DNA + 2 Dye (6X) + 7 Milli-Q H2O
98 - N term tag (B2)1 DNA + 2 Dye (6X) + 7 Milli-Q H2O
109 - pSB NEYFP (B4)1 DNA + 2 Dye (6X) + 7 Milli-Q H2O
1110 - pSB NEYFP (B5)1 DNA + 2 Dye (6X) + 7 Milli-Q H2O
1211 - CFP (B6)1 DNA + 2 Dye (6X) + 7 Milli-Q H2O
1312 - pBAD-TetR (B10)1 DNA + 2 Dye (6X) + 7 Milli-Q H2O
1413 - D31 DNA + 2 Dye (6X) + 7 Milli-Q H2O
1514 - C term (D4)1 DNA + 2 Dye (6X) + 7 Milli-Q H2O
1615 - C term (D5)1 DNA + 2 Dye (6X) + 7 Milli-Q H2O
1716 - pLacI (D6)1 DNA + 2 Dye (6X) + 7 Milli-Q H2O
1817 - NEYFP (E2)1 DNA + 2 Dye (6X) + 7 Milli-Q H2O
1918 - CEYFP (E6)1 DNA + 2 Dye (6X) + 7 Milli-Q H2O
2019 - CEYFP (E7)1 DNA + 2 Dye (6X) + 7 Milli-Q H2O
11kb ladder0.5 Ladder + 2 Dye (6X) + 7.5 Milli-Q H2O
220 - EYFP (E8)1 DNA + 2 Dye (6X) + 7 Milli-Q H2O
321 - EYFP (E9)1 DNA + 2 Dye (6X) + 7 Milli-Q H2O
422 - EYFP (E10)1 DNA + 2 Dye (6X) + 7 Milli-Q H2O
523 - ECFP (F1)1 DNA + 2 Dye (6X) + 7 Milli-Q H2O
624 - ECFP (F2)1 DNA + 2 Dye (6X) + 7 Milli-Q H2O
725 - ECFP (F3)1 DNA + 2 Dye (6X) + 7 Milli-Q H2O
826 - pBAD-TetR (F4)1 DNA + 2 Dye (6X) + 7 Milli-Q H2O
927 - pBAD-TetR (F5)1 DNA + 2 Dye (6X) + 7 Milli-Q H2O
1028 - EYFP (G1)1 DNA + 2 Dye (6X) + 7 Milli-Q H2O
1129 - pSB CEYFP (G4)1 DNA + 2 Dye (6X) + 7 Milli-Q H2O
1230 - pBAD (1) (G6)1 DNA + 2 Dye (6X) + 7 Milli-Q H2O
1331 - pBAD (2) (G7)1 DNA + 2 Dye (6X) + 7 Milli-Q H2O
1432 - N term tag (G8)1 DNA + 2 Dye (6X) + 7 Milli-Q H2O
1533 - 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)
00.071
10.390
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.

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:

LaneSampleVolume
Sample (µL)
Volume Loading
Dye (µL)
1Restricted RBS-xylE102
1Unestricted RBS-xylE12
11kb Ladder22

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

100602 JV rbs-xylE.JPG

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:
    ComponentVolume (µL)
    MilliQ H2O15.5
    Buffer2
    pDNA2
    Enzyme0.25 + 0.25

    Set up control reaction as follows:

    • MilliQ H2O - 16µL
    • Buffer - 2µL
    • pDNA - 2µL

    Incubated reactions for 65 minutes at 37oC
    Killed enzymes by incubating reactions for 10 minutes at 65oC

  • 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 80oC</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 NameLocationConcentration (ng/µL)Volume/rxn (µL)
    pLacI MaxiprepA9990~1
    pLacI (B1)A6440~2
    sRBS-Lum-dT (2)A1965~1
    sRBS-Lum-dT (1)A21145~1
    sRBS-Lum-dT MaxiprepB84780~.2
    sRBS-Lum-dTB74375~.25
    sRBS-Lum-dT (1)G2335~3
    sRBS-Lum-dT (2)G3965~2
    • 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)
    EnzymesTotal Volume
    sRBS-Lum-dT (A1)965143.550.25µL XbaI
    0.25µL PstI
    50
    sRBS-Lum-dT (A2)1145143.550.25µL XbaI
    0.25µL PstI
    50
    pLacI Maxiprep (A1)990143.550.25µL EcoRI
    0.25µL SpeI
    50
    sRBS-Lum-dT Maxiprep(B8)47802 (of 1:10 dilution)42.550.25µL XbaI
    0.25µL PstI
    50
    sRBS-Lum-dT (B7)43752.5 (of 1:10 dilution)4250.25µL XbaI
    0.25µL PstI
    50
    pLacI (D6)440242.550.25µL EcoRI
    0.25µL SpeI
    50
    sRBS-Lum-dT (G2)335341.550.25µL XbaI
    0.25µL PstI
    50
    sRBS-Lum-dT (G3)540242.550.25µL XbaI
    0.25µL PstI
    50
    pSB1T32512.5750.25µL EcoRI
    0.25µL PstI
    50

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