Construction

From 2006.igem.org

Revision as of 02:08, 6 October 2006

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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October 3, 2006

Charles:

• Miniprepped I12006 EFG (2005)
• Made freezer stock of P0456 (2005)
• Replated P0456 (2005) from o/n
• Made o/n of UT4 ABC, UT5 ABC for miniprep and UT2/UT3 for microscope testing

To-Do:

• Miniprep UT4/UT5 ABC and check lengths
• If parts check out, digest E0240, I12006 and UT4/UT5 to prepare for ligation
• Transform UT2 and UT3 into DH5a cells to prepare for temperature testing
• Do another IPTG dose response and try to view cells using fluorescent microscope

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October 2, 2006

Charles, Elliott, Seema, Ting:

• Made competent DH5a-z1 and DH5a
• Made o/n I12006 EFG (2005) for mini-prep
• Made o/n P0456 (2006) for freezer stock (maybe mini-prep to check length)
• Plated UT4 ABC and UT5 ABC

To-Do List:

• Miniprep I12006 EFG (2005)
• Plate I12006 (2006) again
• Make o/n of UT4 and UT5 for mini-prep

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October 1, 2006

Charles, Andy, Anne:

• Prepared o/n of DH5a and DH5a-z1 for making competent cells
• Mini-prepped UT3 ABCD and P0352 (2005) BC, P0456 (2005) BC
• Tested (EcoRI, PstI) length of UT3 ABCD (All wrong lengths, similar to Sept 28 result), P0352 (2005) BC (both have only one band, retry with higher DNA conc.), P0456 (2005) BC (correct lengths)
• Made Plates and transformed P0452 (2006) and I12006 (2006)
• Printed/posted protocols
• Ligated UT1 ABC to P0352 A (2005) and P0456 A (2005) overnight

To-Do List:

• Finish up ligation of UT1 and P0352 and P0456 (Heat inactivate)
• Transform and plate UT4 (UT1 + P0352 (2005)) ABC and UT5 (UT1 + P0456 (2005)) ABC
• Make o/n of P0452 (2006) and I12006 (2006) for miniprep
• Make DH5a and DH5a-z1 competent cells
• Plan testing using DH5a cells

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September 30, 2006

Charles:

• Prepare o/n of UT3 ABCD and P0352, P0456 (2005) (2 vials each)

To-Do List:

• Prepare o/n of DH5a and DH5a-z1 for making competent cells
• Miniprep UT3 ABCD and P0352, P0456 (2005) and test
• Make Plates and transform P0452 (2006) and I12006 (2006)
• Double Digest (total volume = 30 uL) P0352 and P0456 (2005) with EcoI/XbaI in Buffer 2 and then do a gel extract/purification
• Ligate UT1 and P0352/P0456 (3 tries each)

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September 29, 2006

Charles:

• Prepared o/n of DH5a-z1 and UT3 ABCD to miniprep again
• Plated DH5a

To-Do List:

• Make new competent DH5a-z1
• Prepare o/n of regular DH5a
• Plated P0 (2006) using new competent cells
• Double digest UT1 (host) with SpeI/PstI in Buffer 2 and double digest P0352/P0456 (2005) with XbaI/PstI in Buffer 3

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September 28, 2006

Andy:

• Miniprep P0456 (2005)
• Checked the lengths (EcoRI and PstI, Buffer EcoRI): UT3 ABCDE, P0456 (2005) and P0352 (2005)
• Transformed P0152, P0352, P0452, and P0456 (2006). [Plated on Amp plates, because no more Amp/Kan plate]

To-Do List:

• Plate DH5a cells
• Make LB Broth, plates and print protocols
• Digest P0352 and P0456 (2005) and ligate with UT1
• Prepare o/n of UT3 ABCDE again and try to miniprep again

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September 27, 2006

Charles:

• Made freezer stocks of UT2 x 2 and UT3 x 2

Andy, Natalie:

• Mini-preppred UT3 B(AB) C(BB) D(BC) E(CC)
• Transformed and plated P0452 (2005) (On Amp rather than proper AK)
• Checked lengths of UT3 BCDE (inconclusive due to anomalous bands, D appears promising - yellow rating), P0152 (2nd check, Extra band, wrong insert length), P0352 (2nd check, not the best bands, correct lengths - changed rating from red to yellow)
• Prepared o/n of P0456 (2005) for Miniprep
• Added part lengths and resistance info in spreadsheet

To-Do List:

• Miniprep P0456 (2005)
• Take P0456, P0452, P0152, P0352 from 2006 registry
• Check lengths of UT3 BCDE and P0352 again
• Make more plates, make more LB
• Learn to use new DH5a (with no LacI expression) for testing
• Post up new protocols

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