Construction

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<font size=4>< [[Aug11-Sep7]] | [[Sep8-14]] | [[Sep15-26]] | [[Sep27-Oct3]] | [[Oct4-Oct13]] | [[Construction]] ></font>
<font size=4>< [[Aug11-Sep7]] | [[Sep8-14]] | [[Sep15-26]] | [[Sep27-Oct3]] | [[Oct4-Oct13]] | [[Construction]] ></font>
</center>
</center>
 +
 +
== October 19, 2006 ==
 +
 +
'''Melinda:'''
 +
<ul>
 +
    <li>Miniprepped Q04400 (2005/2006) and Q04510 (2005/2006)
 +
    <li>Made o/n of Q03400 (2005/2006), (3) UT2-DH5a, (2) DH5a, and (1) DH5a/DH5a-z1 for freezer stock and fresh plate
 +
</ul>
 +
 +
'''To-Do List:'''
 +
<ul>
 +
    <li>Miniprep Q03400 (2005/2006)
 +
    <li>Repeat Oct 18 test, except with GFP and proper controls
 +
    <li>Digest and check J04450 (W), Q03400, Q04400, and Q04510 (2005/2006).  If correct, move on with gel extraction and ligation.
 +
    <li>Make 20% Arabinose, and 2 blank agar plates
 +
</ul>
 +
 +
[http://2006.igem.org/University_of_Toronto_2006 Home]
== October 18, 2006 ==
== October 18, 2006 ==
Line 22: Line 40:
         so, digest with EcoRI/XbaI and gel extract
         so, digest with EcoRI/XbaI and gel extract
     <li>Ligate with UT1
     <li>Ligate with UT1
 +
</ul>
 +
 +
[http://2006.igem.org/University_of_Toronto_2006 Home]
 +
 +
== October 17, 2006 ==
 +
 +
'''Melinda:'''
 +
<ul>
 +
    <li>Double Digetsed J04450 (W), UT1 DEFG with XbaI/SpeI using Buffer 2
 +
    <li>Results:
 +
    <ul>
 +
        <li>J04450 (W) (1 band)
 +
        <li>UT1 D (5000 – 4000, 3000 – 2500)
 +
        <li>UT1 E (2 bands)
 +
        <li>UT1 F (2 bands)
 +
        <li>UT1 G (2 bands)
 +
    </ul>
 +
    <li>Transformed DH5a cells with Q04400 (2005/2006), Q04510 (2005/2006)
 +
    <li>Made o/n of DH5a and (3 vials) of DH5a UT3
 +
</ul>
 +
 +
'''Note – Change in protocol:'''
 +
<ul>
 +
    <li>After incubation for 1 hour, centrifuge tubes for 5 min @ 3000 RPM to pellet cells
 +
    <li>Remove 800 uL of supernatant
 +
    <li>Resuspend pellet with remaining 200 uL
 +
    <li>Spread on plate and wait ~15-20 min.
 +
</ul>
 +
 +
[http://2006.igem.org/University_of_Toronto_2006 Home]
 +
 +
== October 16, 2006 ==
 +
 +
'''Melinda:'''
 +
<ul>
 +
    <li>Checked parts, including the relevant parts from Waterloo (W):
 +
    <ul>
 +
        <li>I0500 (W): (5000 – 4000, 1500 – 1000) – correct!
 +
        <li>J04450 (W): (2500 – 2000) – plasmid is correct, but no part?
 +
        <li>J06801 (W): (8000 – 6000) – not correct!
 +
        <li>E0240 (W): (4000 – 3500, 3500 – 3000, 1000 – 750) – part is correct?
 +
        <li>I12006 (W): (5000 – 4000, 2500 – 2000)  - plasmid is correct, undigested plasmid?
 +
        <li>UT2 2: (3500 – 3000, 2500 – 2000, 2000 – 1500) – correct, extra band?
 +
        <li>UT2 4: (3500 – 3000, 2500 – 2000, 2000 – 1500) – correct, extra band?
 +
        <li>UT3 7: (3500 – 3000, 2500 – 2000) – correct!
 +
    <li>Viewed cells induced by IPTG under microscope and verified the functionality of UT2/UT3 in DH5a and DH5a-z1
 +
</ul>
 +
 +
'''To-Do List:'''
 +
<ul>
 +
    <li>Repeat Double Digest of J04450 (W) and UT1 ABCD with XbaI/SpeI (Run undigested plasmids beside each double digest)
 +
    <li>Make more Amp plates
 +
    <li>If digest is successful, transform DH5a cells with the successful parts
 +
    <li>Find tetR replacements for cI and ligate those parts together.
 +
</ul>
 +
 +
[http://2006.igem.org/University_of_Toronto_2006 Home]
 +
 +
== October 15, 2006 ==
 +
 +
'''Charles:'''
 +
<ul>
 +
    <li>Made o/n of the newly plated UT2 2/4 and UT3 7 as well as UT2/UT3 freezer stock
 +
</ul>
 +
 +
'''To-Do List:'''
 +
<ul>
 +
    <li>Make sure the cells fluoresce by diluting in IPTG
</ul>
</ul>

Revision as of 03:38, 20 October 2006

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

Contents

October 19, 2006

Melinda:

  • Miniprepped Q04400 (2005/2006) and Q04510 (2005/2006)
  • Made o/n of Q03400 (2005/2006), (3) UT2-DH5a, (2) DH5a, and (1) DH5a/DH5a-z1 for freezer stock and fresh plate

To-Do List:

  • Miniprep Q03400 (2005/2006)
  • Repeat Oct 18 test, except with GFP and proper controls
  • Digest and check J04450 (W), Q03400, Q04400, and Q04510 (2005/2006). If correct, move on with gel extraction and ligation.
  • Make 20% Arabinose, and 2 blank agar plates

Home

October 18, 2006

Andy, Konstantin:

  • Transformed and plated Q03400 (2005) (2006)
  • Gel-extracted UT1 DEF with EcoRI/SpeI (2500-3000, 5000, 8000), 8000 band bright, digestion appeared incomplete
  • Made A, K, AK plates
  • Made o/n of Q04400 (2005) (2006) Q04510 (2005) (2006)
  • Tested UT3 in DH5a with 0%-2% arabinose

To-Do List:

  • Make o/n of Q03400 (2005) (2006)
  • Miniprep Q04400 (2005) (2006) and Q04510 (2005) (2006)
  • Replate UT1 with miniprep from a good conlony
  • Miniprep Q04400 (2005) (2006) Q04510 (2005) (2006) and check to see if bands are correct. If so, digest with EcoRI/XbaI and gel extract
  • Ligate with UT1

Home

October 17, 2006

Melinda:

  • Double Digetsed J04450 (W), UT1 DEFG with XbaI/SpeI using Buffer 2
  • Results:
    • J04450 (W) (1 band)
    • UT1 D (5000 – 4000, 3000 – 2500)
    • UT1 E (2 bands)
    • UT1 F (2 bands)
    • UT1 G (2 bands)
  • Transformed DH5a cells with Q04400 (2005/2006), Q04510 (2005/2006)
  • Made o/n of DH5a and (3 vials) of DH5a UT3

Note – Change in protocol:

  • After incubation for 1 hour, centrifuge tubes for 5 min @ 3000 RPM to pellet cells
  • Remove 800 uL of supernatant
  • Resuspend pellet with remaining 200 uL
  • Spread on plate and wait ~15-20 min.

Home

October 16, 2006

Melinda:

  • Checked parts, including the relevant parts from Waterloo (W):
    • I0500 (W): (5000 – 4000, 1500 – 1000) – correct!
    • J04450 (W): (2500 – 2000) – plasmid is correct, but no part?
    • J06801 (W): (8000 – 6000) – not correct!
    • E0240 (W): (4000 – 3500, 3500 – 3000, 1000 – 750) – part is correct?
    • I12006 (W): (5000 – 4000, 2500 – 2000) - plasmid is correct, undigested plasmid?
    • UT2 2: (3500 – 3000, 2500 – 2000, 2000 – 1500) – correct, extra band?
    • UT2 4: (3500 – 3000, 2500 – 2000, 2000 – 1500) – correct, extra band?
    • UT3 7: (3500 – 3000, 2500 – 2000) – correct!
    • Viewed cells induced by IPTG under microscope and verified the functionality of UT2/UT3 in DH5a and DH5a-z1

    To-Do List:

    • Repeat Double Digest of J04450 (W) and UT1 ABCD with XbaI/SpeI (Run undigested plasmids beside each double digest)
    • Make more Amp plates
    • If digest is successful, transform DH5a cells with the successful parts
    • Find tetR replacements for cI and ligate those parts together.

    Home

    October 15, 2006

    Charles:

    • Made o/n of the newly plated UT2 2/4 and UT3 7 as well as UT2/UT3 freezer stock

    To-Do List:

    • Make sure the cells fluoresce by diluting in IPTG

    Home

    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+
    Home
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