Construction

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

Contents

October 12, 2006

Andy, Charles, Melinda, Natalie, Ram, Siva, Tara:

  • Made new agar plates (amp = 5, kan = 5, amp/kan = 5)
  • Made o/n of UT2 (5), UT3 (5) and DH5a
      1. Plate BA, colony 20
      2. Plate BB, colony 21
      3. Plate CA, colony 22
      4. Plate CA, colony 23
      5. Plate CB, colony 24
      1. Plate AB, colony 25
      2. Plate BB, colony 26
      3. Plate BB, colony 27
      4. Plate BC, colony 28
      5. Plate CC, colony 29
    • Also made o/n for UT2 (2), UT3 (2) from freezer stock
  • Transformed I12006 AB (2005)
  • Digested and checked lengths
    • I12006 ABH (2005): (5000 – 4000) – correct
    • UT1 AB: (5000 – 4000, 3500 – 3000) – correct
    • UT2 EF (3000 – 2500, 1000 – 750) – not so correct
  • Digested I12006 AB (2005) with SpeI/PstI and E0240 EF (2005) with XbaI/PstI
    • I12006 ABH (2005): (5000 – 4000, 2000 – 1500) – not so correct
    • E0240 EF (2005): (3500 – 3000, 2500 – 2000, 2000 – 1500, 1000 – 750) – not correct
    • Did not gel extract I12006 and E0240

To-Do List:

  • Try different ligation enzymes, thus ligate UT4 with I12006 then ligate with E0240.
    • Digest I12006 AB (2005) with EcoRI/XbaI (host) and UT4 AB with EcoRI/SpeI (insert)
  • Check UT2/UT3 under microscope to find out if we have fluorescence
    • If we do, mini-prep the o/n and transform cells
  • DH5a vs. [Arabinose] to test potential arabinose induced fluorescence reduction
    • Use PBS + Arabinose as a reference

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October 11, 2006

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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