Team:Lethbridge/Notebook/Lab Work/June

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Incubated reactions overnight at room temperature (total of 19.5 hours)<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>
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>
 +
<ol>
 +
<li>Want a final mass of 25ng of each pDNA in the ligation mix.
 +
<li>Final concentration of pDNA in restriction digest should be 25-50ng/&micro;L.
 +
<li>Tom Knight's restriction reaction is 50&micro;L, therefore there should be 1000ng pDNA in each restriction digest.
 +
<li>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>
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<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.
 +
<li>Cut pLacI with EcoRI and SpeI
 +
<li>Cut sRBS-Lum-dT with XbaI and PstI
 +
<li>Cut pSB1T3 with EcoRI and PstI
 +
<li>Will have total of 12 ligation reactions, want 12x2&micro;L of pSB1T3 to add to each, therefore want 25&micro;L of pSB1T3.</ol>
 +
<b>Method:</b><br>
 +
Experimental Setup<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 (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 (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 (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 (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 (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 (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 (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 (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></table>
 +
===June 3/2010===
===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>
<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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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-xylE†12
11kb Ladder††22

† 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:

    1. Want a final mass of 25ng of each pDNA in the ligation mix.
    2. Final concentration of pDNA in restriction digest should be 25-50ng/µL.
    3. Tom Knight's restriction reaction is 50µL, therefore there should be 1000ng pDNA in each restriction digest.
    4. 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.
    5. Cut pLacI with EcoRI and SpeI
    6. Cut sRBS-Lum-dT with XbaI and PstI
    7. Cut pSB1T3 with EcoRI and PstI
    8. Will have total of 12 ligation reactions, want 12x2µL of pSB1T3 to add to each, therefore want 25µL of pSB1T3.</ol> Method:
      Experimental Setup
      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 (A1)965143.550.25µL XbaI
      0.25µL PstI      		50
      sRBS-Lum-dT (A1)965143.550.25µL XbaI
      0.25µL PstI      		50
      sRBS-Lum-dT (A1)965143.550.25µL XbaI
      0.25µL PstI      		50
      sRBS-Lum-dT (A1)965143.550.25µL XbaI
      0.25µL PstI      		50
      sRBS-Lum-dT (A1)965143.550.25µL XbaI
      0.25µL PstI      		50
      sRBS-Lum-dT (A1)965143.550.25µL XbaI
      0.25µL PstI      		50
      sRBS-Lum-dT (A1)965143.550.25µL XbaI
      0.25µL PstI      		50
      sRBS-Lum-dT (A1)965143.550.25µL XbaI
      0.25µL PstI      		50


      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.
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