Construction

From 2006.igem.org

Revision as of 04:10, 2 October 2006

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, same as Sept ## result), P0352 (2005) BC (both have only one band, retry with higher conc. DNA), 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
• Miniprep

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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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