Construction

From 2006.igem.org

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

Contents 1 October 21, 2006 2 October 19, 2006 3 October 18, 2006 4 October 17, 2006 5 October 16, 2006 6 October 15, 2006 7 October 14, 2006

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

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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+

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