Construction

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<font size=4>< [[Aug11-Sep7]] | [[Sep8-14]] | [[Sep15-26]] | [[Sep27-Oct3]] | [[Construction]] ></font>
<font size=4>< [[Aug11-Sep7]] | [[Sep8-14]] | [[Sep15-26]] | [[Sep27-Oct3]] | [[Construction]] ></font>
</center>
</center>
 +
 +
    <li>Digest I12006 AB (2005) with EcoRI/XbaI (host) and UT4 AB with EcoRI/SpeI (insert)
 +
        <ul>
 +
            <li>Ligate I12006 and UT4
 +
        </ul>
 +
== October 11, 2006 ==
== October 11, 2006 ==
 +
 +
Long-Term Goals:
 +
- Once the mini-preps of the correct lengths are obtained, retest UT2/UT3 with increasing IPTG to repeat previous experiment (if successful, take pictures)
 +
- Need to test to see if arabinose is having deleterious effects on fluorescence
 +
o DH5a auto-fluorescence vs. [Arabinose]
 +
o Consider using a tetR promoter instead of pBad/araC
 +
- Do an Arabinose and IPTG surface response test to find optimal concentrations
 +
- NOTE: try not to overgrow the cells – signal seems to saturate quickly
 +
'''To-Do List:'''
'''To-Do List:'''
<ul>
<ul>
-
     <li>Digest I12006 H (2005) and I12006 AB (2006) with XbaI/SpeI
+
     <li>Digest I12006 H (2005) and I12006 AB (2006) with XbaI/SpeI and check lengths
     <li>Transform cells with I12006 AB (2005)
     <li>Transform cells with I12006 AB (2005)
     <li>Transform cells with UT1 AB, UT2 EF, UT4 AB
     <li>Transform cells with UT1 AB, UT2 EF, UT4 AB
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             <li>Try to find the UT4 used to make a measurement (that was successful)
             <li>Try to find the UT4 used to make a measurement (that was successful)
         </ul>
         </ul>
-
     <li>Digest I12006 AB (2005) with EcoRI/XbaI (host) and UT4 AB with EcoRI/SpeI (insert)
+
     <li>Digest I12006 AB (2005) with SpeI/PstI
         <ul>
         <ul>
-
             <li>Ligate I12006 and UT4
+
             <li>Ligate I12006 AB (2005) and E0240 (purified DNA from Oct4)
         </ul>
         </ul>
 +
</ul>
 +
 +
'''Long-Term Goals:'''
 +
<ul>
 +
    <li>Once the mini-preps of the correct lengths are obtained, retest UT2/UT3 with increasing IPTG
 +
        to repeat previous experiment (if successful, take pictures)
     <li>Need to test to see if arabinose is having deleterious effects on fluorescence
     <li>Need to test to see if arabinose is having deleterious effects on fluorescence
         <ul>
         <ul>
Line 22: Line 43:
             <li>Consider using a tetR promoter instead of pBad/araC
             <li>Consider using a tetR promoter instead of pBad/araC
         </ul>
         </ul>
-
     <li>Once the mini-preps of the correct lengths are obtained, retest UT2/UT3 with increasing IPTG to repeat previous experiment (if
+
     <li>Do an Arabinose and IPTG surface response test to find optimal concentrations
-
        successful, take pictures)
+
    <li>NOTE: try not to overgrow the cells – signal seems to saturate quickly
</ul>
</ul>

Revision as of 04:45, 13 October 2006

< Aug11-Sep7 | Sep8-14 | Sep15-26 | Sep27-Oct3 | Construction >

  • Digest I12006 AB (2005) with EcoRI/XbaI (host) and UT4 AB with EcoRI/SpeI (insert)
    • Ligate I12006 and UT4


    Contents

    October 11, 2006

    Long-Term Goals: - Once the mini-preps of the correct lengths are obtained, retest UT2/UT3 with increasing IPTG to repeat previous experiment (if successful, take pictures) - Need to test to see if arabinose is having deleterious effects on fluorescence o DH5a auto-fluorescence vs. [Arabinose] o Consider using a tetR promoter instead of pBad/araC - Do an Arabinose and IPTG surface response test to find optimal concentrations - NOTE: try not to overgrow the cells – signal seems to saturate quickly


    To-Do List:

    • Digest I12006 H (2005) and I12006 AB (2006) with XbaI/SpeI and check lengths
    • Transform cells with I12006 AB (2005)
    • Transform cells with UT1 AB, UT2 EF, UT4 AB
      • Try to find the UT4 used to make a measurement (that was successful)
    • Digest I12006 AB (2005) with SpeI/PstI
      • Ligate I12006 AB (2005) and E0240 (purified DNA from Oct4)

    Long-Term Goals:

    • Once the mini-preps of the correct lengths are obtained, retest UT2/UT3 with increasing IPTG to repeat previous experiment (if successful, take pictures)
    • Need to test to see if arabinose is having deleterious effects on fluorescence
      • DH5a auto-fluorescence vs. [Arabinose]
      • Consider using a tetR promoter instead of pBad/araC
    • Do an Arabinose and IPTG surface response test to find optimal concentrations
    • NOTE: try not to overgrow the cells – signal seems to saturate quickly

    [http://2006.igem.org/University_of_Toronto_2006 Home]

    October 8, 2006

    Anne:

    • Prepared DH5a UT2 for LacI temperature test
    • Mini-prepped I2006 H (2005) and I12006 AB (2006)

    [http://2006.igem.org/University_of_Toronto_2006 Home]

    October 7, 2006

    Cheng:

    • Prepared DH5a-z1 UT2 for IPTG induction of GFP

    Conrad:

    • Re-test UT4C, UT5 ABC and I12006 EFG (2005) with XbaI/SpeI
    • Miniprep UT2/3 in DH5a

    Siva:

    • Prepared DH5a UT2 for Arabinose induced LacI test

    Recorded digest length check results:

    • I12006 EFG (4000-5000, 1500-1000)
    • UT5 ABC (3500-4000, 750-1000)
    • UT4 C (3000-3500)
    • UT3 DH5a (750-1000, 2000-2500)
    • UT2 DH5a (750-1000, 2000-2500)

    Test Results

    [http://2006.igem.org/University_of_Toronto_2006 Home]

    October 6, 2006

    Andy, HoKwon:

    • Prepared o/n (3 vials each) of DH5a and DH5a-z1 with UT2 and UT3 for testing
    • Prepared o/n of UT2 DH5a and UT3 DH5a for miniprep
    • Transformed and plated I12006 (2005) (poured in amp plate first by accident), I12006 (2006)
    • Double Digest parts E0240 CD (2005), UT4 AB with XbaI/SpeI
      • E0240 CD (2000-2500, 750-1000) Correct!
      • UT4 AB (3000-4000) Correct!

    Charles, Cheng, Nick:

    • Re-tested UT4 C, UT5 ABC – failed due to a very dry gel

    [http://2006.igem.org/University_of_Toronto_2006 Home]

    October 4, 2006

    Andy:

    • Checked lengths (EcoRI, PstI) of following
      • I12006 EFG (4000-5000, 1000-1500 for all three)
      • UT4 AB (3000-4000, 2000) C (3000-4000, 250)
      • UT5 ABC (2500-3000, 750-1000)
    • Digested following
      • I12006 (2005) E (6000 by SpeI and PstI)
      • E0240 (2005) C (2500-3000, 2000-2500, 1500-2000, 750-1000 by XbaI and PstI)
      • UT4 AB (6000-10000, 2000-2500 by SpeI and PstI) C (5000-6000, 3000 by SpeI and PstI)
      • UT5 ABC (3000-4000 by SpeI and PstI)
    • Gel extracted E0240 (insert) and UT4 AB (host)
    • Transformed and plated DH5a with UT2/UT3 for testing
    • Made o/n of UT2/UT3 with IPTG

    To-Do:

    • Redo transformation of I12006 (2005)
    • Learn to take fluorescent images under microscope
    • Temperature testing
    • Try ligating UT5 again
    • Running out of water and PCR tubes

    [http://2006.igem.org/University_of_Toronto_2006 Home]

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