Construction

• Ligate I12006 and UT4

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

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October 8, 2006

Anne:

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

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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

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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

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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

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