Construction

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<font size=4>< [[Aug11-Sep7]] | [[Sep8-14]] | [[Sep15-26]] | [[Sep27-Oct3]] | [[Construction]] ></font>
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<font size=4>< [[Aug11-Sep7]] | [[Sep8-14]] | [[Sep15-26]] | [[Sep27-Oct3]] | [[Oct4-Oct13]] | [[Construction]] ></font>
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     <li>Make o/n of working UT2/UT3 for repeat of IPTG test.
     <li>Make o/n of working UT2/UT3 for repeat of IPTG test.
     <li>Look into using tetR and tet pL instead of cI and Prm+
     <li>Look into using tetR and tet pL instead of cI and Prm+
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</ul>
 
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[http://2006.igem.org/University_of_Toronto_2006 Home]
 
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== October 13, 2006 ==
 
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'''Charles, Jovan, Nick:'''
 
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<ul>
 
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    <li>Prepared tubes at 1:20 dilution of UT2/UT3 o/n for fluorescence at 2000 uM IPTG
 
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    <li>Prepared tubes at 1:20 dilution of DH5a o/n for LacI repression at 0%, 0.02%, 0.2% and 2%
 
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        Arabinose
 
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    <li>Digested I12006 ABCD (2005), I12006 AB (2006) with EcoRI/SpeI (which was a mistake)
 
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    <li>Checked UT2/UT3 and we found the original glowing colonies (UT2 1/2 and UT3 7).
 
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    <ul>
 
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        <li>UT2 (2) Plate BB, colony 21 and (4) Plate CA, colony 23
 
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        <li>UT3 (7) Plate BB, colony 26
 
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        <li>Couldn’t take pictures due time constraints
 
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    </ul>
 
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</ul>
 
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'''To-Do List:'''
 
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<ul>
 
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    <li>Verify last day’s results through fluorescence microscopy and mini-prep and transform DH5a-z1
 
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        and DH5a
 
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    <li>Wait for Natalie to bring the biobricks from Waterloo to obtain I12006 and J04450
 
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</ul>
 
-
 
-
[http://2006.igem.org/University_of_Toronto_2006 Home]
 
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== October 12, 2006 ==
 
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'''Andy, Charles, Melinda, Natalie, Ram, Siva, Tara:'''
 
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<ul>
 
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    <li>Made new agar plates (amp = 5, kan = 5, amp/kan = 5)
 
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    <li>Made o/n of UT2 (5), UT3 (5) and DH5a
 
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        <ul>
 
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            <li>UT2:
 
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                <ol>
 
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                    <li>Plate BA, colony 20
 
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                    <li>Plate BB, colony 21
 
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                    <li>Plate CA, colony 22
 
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                    <li>Plate CA, colony 23
 
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                    <li>Plate CB, colony 24
 
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                </ol>
 
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            <li>UT3:
 
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                <ol continue>
 
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                    <li>Plate AB, colony 25
 
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                    <li>Plate BB, colony 26
 
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                    <li>Plate BB, colony 27
 
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                    <li>Plate BC, colony 28
 
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                    <li>Plate CC, colony 29
 
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                </ol>
 
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            <li>Also made o/n for UT2 (2), UT3 (2) from freezer stock
 
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        </ul>
 
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    <li>Transformed I12006 AB (2005)
 
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    <li>Digested and checked lengths
 
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        <ul>
 
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            <li>I12006 ABH (2005): (5000 – 4000) – correct
 
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            <li>UT1 AB: (5000 – 4000, 3500 – 3000) – correct
 
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            <li>UT2 EF (3000 – 2500, 1000 – 750) – not so correct
 
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        </ul>
 
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    <li>Digested I12006 AB (2005) with SpeI/PstI and E0240 EF (2005) with XbaI/PstI
 
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        <ul>
 
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            <li>I12006 ABH (2005): (5000 – 4000, 2000 – 1500) – not so correct
 
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            <li>E0240 EF (2005): (3500 – 3000, 2500 – 2000, 2000 – 1500, 1000 – 750) – not correct
 
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            <li>Did not gel extract I12006 and E0240
 
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        </ul>
 
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</ul>
 
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'''To-Do List:'''
 
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<ul>
 
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    <li>Try different ligation enzymes, thus ligate UT4 with I12006 then ligate with E0240.
 
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        <ul>
 
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            <li>Digest I12006 AB (2005) with EcoRI/XbaI (host) and UT4 AB with EcoRI/SpeI (insert)
 
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        </ul>
 
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    <li>Check UT2/UT3 under microscope to find out if we have fluorescence
 
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        <ul>
 
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            <li>If we do, mini-prep the o/n and transform cells
 
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        </ul>
 
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    <li>DH5a vs. [Arabinose] to test potential arabinose induced fluorescence reduction
 
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        <ul>
 
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            <li>Use PBS + Arabinose as a reference
 
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        </ul>
 
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</ul>
 
-
 
-
[http://2006.igem.org/University_of_Toronto_2006 Home]
 
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== October 11, 2006 ==
 
-
 
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'''To-Do List:'''
 
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<ul>
 
-
    <li>Digest I12006 H (2005) and I12006 AB (2006) with XbaI/SpeI and check lengths
 
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    <li>Transform cells with I12006 AB (2005)
 
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    <li>Transform cells with UT1 AB, UT2 EF, UT4 AB
 
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        <ul>
 
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            <li>Try to find the UT4 used to make a measurement (that was successful)
 
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        </ul>
 
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    <li>Digest I12006 AB (2005) with SpeI/PstI
 
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        <ul>
 
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            <li>Ligate I12006 AB (2005) and E0240 (purified DNA from Oct4)
 
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        </ul>
 
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</ul>
 
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'''Long-Term Goals:'''
 
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<ul>
 
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    <li>Once the mini-preps of the correct lengths are obtained, retest UT2/UT3 with increasing IPTG
 
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        to repeat previous experiment (if successful, take pictures)
 
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    <li>Need to test to see if arabinose is having deleterious effects on fluorescence
 
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        <ul>
 
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            <li>DH5a auto-fluorescence vs. [Arabinose]
 
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            <li>Consider using a tetR promoter instead of pBad/araC
 
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        </ul>
 
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    <li>Do an Arabinose and IPTG surface response test to find optimal concentrations
 
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    <li>NOTE: try not to overgrow the cells – signal seems to saturate quickly
 
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</ul>
 
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-
[http://2006.igem.org/University_of_Toronto_2006 Home]
 
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== October 8, 2006 ==
 
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'''Anne:'''
 
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<ul>
 
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    <li>Prepared DH5a UT2 for LacI temperature test
 
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    <li>Mini-prepped I2006 H (2005) and I12006 AB (2006)
 
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</ul>
 
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-
[http://2006.igem.org/University_of_Toronto_2006 Home]
 
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== October 7, 2006 ==
 
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'''Cheng:'''
 
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<ul>
 
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    <li>Prepared DH5a-z1 UT2 for IPTG induction of GFP
 
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</ul>
 
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'''Conrad:'''
 
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<ul>
 
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    <li>Re-test UT4C, UT5 ABC and I12006 EFG (2005) with XbaI/SpeI
 
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    <li>Miniprep UT2/3 in DH5a
 
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</ul>
 
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'''Siva:'''
 
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<ul>
 
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    <li>Prepared DH5a UT2 for Arabinose induced LacI test
 
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</ul>
 
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'''Recorded digest length check results:'''
 
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<ul>
 
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    <li>I12006 EFG (4000-5000, 1500-1000)
 
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    <li>UT5 ABC (3500-4000, 750-1000)
 
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    <li>UT4 C (3000-3500)
 
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    <li>UT3 DH5a (750-1000, 2000-2500)
 
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    <li>UT2 DH5a (750-1000, 2000-2500)
 
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</ul>
 
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'''Test Results'''
 
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-
[http://2006.igem.org/University_of_Toronto_2006 Home]
 
-
 
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== October 6, 2006 ==
 
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'''Andy, HoKwon:'''
 
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<ul>
 
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    <li>Prepared o/n (3 vials each) of DH5a and DH5a-z1 with UT2 and UT3 for testing
 
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    <li>Prepared o/n of UT2 DH5a and UT3 DH5a for miniprep
 
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    <li>Transformed and plated I12006 (2005) (poured in amp plate first by accident), I12006 (2006)
 
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    <li>Double Digest parts E0240 CD (2005), UT4 AB with XbaI/SpeI
 
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    <ul>
 
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        <li>E0240 CD (2000-2500, 750-1000) Correct!
 
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        <li>UT4 AB (3000-4000) Correct!
 
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    </ul>
 
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</ul>
 
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'''Charles, Cheng, Nick:'''
 
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<ul>
 
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    <li>Re-tested UT4 C, UT5 ABC – failed due to a very dry gel
 
-
</ul>
 
-
 
-
[http://2006.igem.org/University_of_Toronto_2006 Home]
 
-
 
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== October 4, 2006 ==
 
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'''Andy:'''
 
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<ul>
 
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    <li>Checked lengths (EcoRI, PstI) of following
 
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        <ul>
 
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            <li>I12006 EFG (4000-5000, 1000-1500 for all three)
 
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            <li>UT4 AB (3000-4000, 2000) C (3000-4000, 250)
 
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            <li>UT5 ABC (2500-3000, 750-1000)
 
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        </ul>
 
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    <li>Digested following
 
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        <ul>
 
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            <li>I12006 (2005) E (6000 by SpeI and PstI)
 
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            <li>E0240 (2005) C (2500-3000, 2000-2500, 1500-2000, 750-1000 by XbaI and PstI)
 
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            <li>UT4 AB (6000-10000, 2000-2500 by SpeI and PstI) C (5000-6000, 3000 by SpeI and PstI)
 
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            <li>UT5 ABC (3000-4000 by SpeI and PstI)
 
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        </ul>
 
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    <li>Gel extracted E0240 (insert) and UT4 AB (host)
 
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    <li>Transformed and plated DH5a with UT2/UT3 for testing
 
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    <li>Made o/n of UT2/UT3 with IPTG
 
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</ul>
 
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'''To-Do:'''
 
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<ul>
 
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    <li>Redo transformation of I12006 (2005)
 
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    <li>Learn to take fluorescent images under microscope
 
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    <li>Temperature testing
 
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    <li>Try ligating UT5 again
 
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    <li>Running out of water and PCR tubes
 
</ul>
</ul>
[http://2006.igem.org/University_of_Toronto_2006 Home]
[http://2006.igem.org/University_of_Toronto_2006 Home]

Revision as of 03:12, 16 October 2006

< Aug11-Sep7 | Sep8-14 | Sep15-26 | Sep27-Oct3 | Oct4-Oct13 | Construction >

October 14, 2006

Charles, Jovan:

  • Mini-prepped UT2 2/4 and UT3 7
  • Transform UT2 2/4 and UT3 7 into DH5a-z1 and DH5a cells
  • Continue with the IPTG test for the rest of the colonies that we weren’t able to test yesterday

To-Do List:

  • Measure absorbance spectrum of Arabinose
  • Make o/n of working UT2/UT3 for repeat of IPTG test.
  • Look into using tetR and tet pL instead of cI and Prm+

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