Construction

From 2006.igem.org

Revision as of 00:59, 23 October 2006 by Andyhung (Talk | contribs)
Jump to: navigation, search

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

Contents

October 21, 2006

Massive To-do List

The following is a guideline. Day-to-day details need to be filled in as we progress. Change colors from red to black as each task is completed.

Construction:

  • Gel-extract Q03400, Q04400, Q04510 (2005/2006) minipreps
  • Transform and miniprep I13522 (2006) (amp) from registry DNA-2 15H
  • Transform and miniprep I13504 (2005/2006) (amp) from registry DNA-1 12D (2005/2006)
  • Miniprep 6 UT1 for ligation
  • Ligate UT1 with Q03400, Q04400, Q04510 (2005/2006) (6 ligations) (Name UT6-12)
  • If possible, ligate R0011 (2005) with I13507 (2005) in parallel
  • Ligate UT6-12 with I13504
  • Make 20% Arabinose, and 2 blank agar plates
  • 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

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

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

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

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

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

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

[http://2006.igem.org/University_of_Toronto_2006 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+

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

Personal tools
Past/present/future years