Team:Lethbridge/Notebook/Lab Work/June

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(June 2/2010 - Evening)
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===June 3/2010===
===June 3/2010===
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<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>
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Carried out protocol described in June 2/2010 - Evening<br>
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Analyzed results on 1% agarose gel.Load order as follows:<br>
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<table><table border ="3">
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<tr><td><b>Lane</b></td><td><b>Gel 1<br>Sample</b></td><td><b>Gel 1 Load</b></td><td><b>Gel 2<br>Sample</b></td><td><b>Gel 2 Load</b></td></tr>
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<tr><td>1</td><td>1kb Ladder</td><td>2&micro;L dye, 2&micro;L ladder<br>8&micro;L MilliQ H<sub>2</sub>O</td><td>1kb Ladder</td><td>2&micro;L dye, 2&micro;L ladder<br>8&micro;L MilliQ H<sub>2</sub>O</td></tr>
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<tr><td>2</td><td>Restricted<br>sRBS-Lum-dT (A1)</td><td>10&micro;L DNA<br>2&micro;L Dye</td><td>pSB1T3 Ligation of:<br>pLacI(A9)+sRBS-Lum-dT(A1)</td><td>10&micro;L DNA<br>2&micro;L Dye</td>

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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 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

    June 3/2010

    Carried out protocol described in June 2/2010 - Evening
    Analyzed results on 1% agarose gel.Load order as follows:

    LaneGel 1
    Sample
    Gel 1 LoadGel 2
    Sample
    Gel 2 Load
    11kb Ladder2µL dye, 2µL ladder
    8µL MilliQ H2O
    1kb Ladder2µL dye, 2µL ladder
    8µL MilliQ H2O
    2Restricted
    sRBS-Lum-dT (A1)
    10µL DNA
    2µL Dye
    pSB1T3 Ligation of:
    pLacI(A9)+sRBS-Lum-dT(A1)
    10µL DNA
    2µL Dye