Team:Lethbridge/Notebook/Lab Work/June

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(June 2/2010)
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*MilliQ H<sub>2</sub>O - 1.75&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>
Incubated reactions overnight at room temperature (total of 19.5 hours)<br>
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Killed enzymes by incubating reactions for 10 minutes at 80<sup>o</sup>C<br>
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Killed enzymes by incubating reactions for 10 minutes at 80<sup>o</sup>C</ul>
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</ul>
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===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-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 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.