Oct4-Oct13

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== October 13, 2006 == '''Charles, Jovan, Nick:''' <ul> <li>Prepared tubes at 1:20 dilution of UT2/UT3 o/n for fluorescence at 2000 uM IPTG <li>Prepared tubes at 1:20 dilution of DH5a o/n for LacI repression at 0%, 0.02%, 0.2% and 2% Arabinose <li>Digested I12006 ABCD (2005), I12006 AB (2006) with EcoRI/SpeI (which was a mistake) <li>Checked UT2/UT3 and we found the original glowing colonies (UT2 1/2 and UT3 7). <ul> <li>UT2 (2) Plate BB, colony 21 and (4) Plate CA, colony 23 <li>UT3 (7) Plate BB, colony 26 <li>Couldn’t take pictures due time constraints </ul> </ul> '''To-Do List:''' <ul> <li>Verify last day’s results through fluorescence microscopy and mini-prep and transform DH5a-z1 and DH5a <li>Wait for Natalie to bring the biobricks from Waterloo to obtain I12006 and J04450 </ul> [http://2006.igem.org/Construction Construction Home] == October 12, 2006 == '''Andy, Charles, Melinda, Natalie, Ram, Siva, Tara:''' <ul> <li>Made new agar plates (amp = 5, kan = 5, amp/kan = 5) <li>Made o/n of UT2 (5), UT3 (5) and DH5a <ul> <li>UT2: <ol> <li>Plate BA, colony 20 <li>Plate BB, colony 21 <li>Plate CA, colony 22 <li>Plate CA, colony 23 <li>Plate CB, colony 24 </ol> <li>UT3: <ol continue> <li>Plate AB, colony 25 <li>Plate BB, colony 26 <li>Plate BB, colony 27 <li>Plate BC, colony 28 <li>Plate CC, colony 29 </ol> <li>Also made o/n for UT2 (2), UT3 (2) from freezer stock </ul> <li>Transformed I12006 AB (2005) <li>Digested and checked lengths <ul> <li>I12006 ABH (2005): (5000 – 4000) – correct <li>UT1 AB: (5000 – 4000, 3500 – 3000) – correct <li>UT2 EF (3000 – 2500, 1000 – 750) – not so correct </ul> <li>Digested I12006 AB (2005) with SpeI/PstI and E0240 EF (2005) with XbaI/PstI <ul> <li>I12006 ABH (2005): (5000 – 4000, 2000 – 1500) – not so correct <li>E0240 EF (2005): (3500 – 3000, 2500 – 2000, 2000 – 1500, 1000 – 750) – not correct <li>Did not gel extract I12006 and E0240 </ul> </ul> '''To-Do List:''' <ul> <li>Try different ligation enzymes, thus ligate UT4 with I12006 then ligate with E0240. <ul> <li>Digest I12006 AB (2005) with EcoRI/XbaI (host) and UT4 AB with EcoRI/SpeI (insert) </ul> <li>Check UT2/UT3 under microscope to find out if we have fluorescence <ul> <li>If we do, mini-prep the o/n and transform cells </ul> <li>DH5a vs. [Arabinose] to test potential arabinose induced fluorescence reduction <ul> <li>Use PBS + Arabinose as a reference </ul> </ul> [http://2006.igem.org/Construction Construction Home] == October 11, 2006 == '''To-Do List:''' <ul> <li>Digest I12006 H (2005) and I12006 AB (2006) with XbaI/SpeI and check lengths <li>Transform cells with I12006 AB (2005) <li>Transform cells with UT1 AB, UT2 EF, UT4 AB <ul> <li>Try to find the UT4 used to make a measurement (that was successful) </ul> <li>Digest I12006 AB (2005) with SpeI/PstI <ul> <li>Ligate I12006 AB (2005) and E0240 (purified DNA from Oct4) </ul> </ul> '''Long-Term Goals:''' <ul> <li>Once the mini-preps of the correct lengths are obtained, retest UT2/UT3 with increasing IPTG to repeat previous experiment (if successful, take pictures) <li>Need to test to see if arabinose is having deleterious effects on fluorescence <ul> <li>DH5a auto-fluorescence vs. [Arabinose] <li>Consider using a tetR promoter instead of pBad/araC </ul> <li>Do an Arabinose and IPTG surface response test to find optimal concentrations <li>NOTE: try not to overgrow the cells – signal seems to saturate quickly </ul> [http://2006.igem.org/Construction Construction Home] == October 8, 2006 == '''Anne:''' <ul> <li>Prepared DH5a UT2 for LacI temperature test <li>Mini-prepped I2006 H (2005) and I12006 AB (2006) </ul> [http://2006.igem.org/Construction Construction Home] == October 7, 2006 == '''Cheng:''' <ul> <li>Prepared DH5a-z1 UT2 for IPTG induction of GFP </ul> '''Conrad:''' <ul> <li>Re-test UT4C, UT5 ABC and I12006 EFG (2005) with XbaI/SpeI <li>Miniprep UT2/3 in DH5a </ul> '''Siva:''' <ul> <li>Prepared DH5a UT2 for Arabinose induced LacI test </ul> '''Recorded digest length check results:''' <ul> <li>I12006 EFG (4000-5000, 1500-1000) <li>UT5 ABC (3500-4000, 750-1000) <li>UT4 C (3000-3500) <li>UT3 DH5a (750-1000, 2000-2500) <li>UT2 DH5a (750-1000, 2000-2500) </ul> '''Test Results''' [http://2006.igem.org/Construction Construction Home] == October 6, 2006 == '''Andy, HoKwon:''' <ul> <li>Prepared o/n (3 vials each) of DH5a and DH5a-z1 with UT2 and UT3 for testing <li>Prepared o/n of UT2 DH5a and UT3 DH5a for miniprep <li>Transformed and plated I12006 (2005) (poured in amp plate first by accident), I12006 (2006) <li>Double Digest parts E0240 CD (2005), UT4 AB with XbaI/SpeI <ul> <li>E0240 CD (2000-2500, 750-1000) Correct! <li>UT4 AB (3000-4000) Correct! </ul> </ul> '''Charles, Cheng, Nick:''' <ul> <li>Re-tested UT4 C, UT5 ABC – failed due to a very dry gel </ul> [http://2006.igem.org/University_of_Toronto_2006 Home] == October 4, 2006 == '''Andy:''' <ul> <li>Checked lengths (EcoRI, PstI) of following <ul> <li>I12006 EFG (4000-5000, 1000-1500 for all three) <li>UT4 AB (3000-4000, 2000) C (3000-4000, 250) <li>UT5 ABC (2500-3000, 750-1000) </ul> <li>Digested following <ul> <li>I12006 (2005) E (6000 by SpeI and PstI) <li>E0240 (2005) C (2500-3000, 2000-2500, 1500-2000, 750-1000 by XbaI and PstI) <li>UT4 AB (6000-10000, 2000-2500 by SpeI and PstI) C (5000-6000, 3000 by SpeI and PstI) <li>UT5 ABC (3000-4000 by SpeI and PstI) </ul> <li>Gel extracted E0240 (insert) and UT4 AB (host) <li>Transformed and plated DH5a with UT2/UT3 for testing <li>Made o/n of UT2/UT3 with IPTG </ul> '''To-Do:''' <ul> <li>Redo transformation of I12006 (2005) <li>Learn to take fluorescent images under microscope <li>Temperature testing <li>Try ligating UT5 again <li>Running out of water and PCR tubes </ul> [http://2006.igem.org/Construction Construction Home]

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