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Team:Nevada/Notebook
From 2009.igem.org
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June 8, 2009 to June 15, 2009
Janice/Leigh: Made LB agar plates and liquid LB with tetracycline. Made tetracycline stock solution (5mg/ml).
June 16, 2009 to June 23, 2009
Janice/Leigh: Made LB liquid media and performed QIAGEN Plasmid Maxi Preps for 1) BBa_B0014 in pSB1AK3 (double terminator), 2) BBA_B0034 in pSB1A3 (ribosome binding site), 3) BBA_R0011 in pSB1A3 (lac I promoter), and 4) BBa_I0500 in pSB2K3 (pBAD/Arac promoter). Made glycerol stocks for 1) to 4). Minipreps were done on the following Arabidopsis genes: 1) cinnamoyl-CoA reductase (CCR2), 2) cinnamoyl-CoA reductase (CCR1), 3) phenylalanine-ammonia lyase, 4) 4-coumarate:CoA ligase 1, and 5) 4-coumarate:CoA ligase 2. Cinnamoyl-CoA reductase (CCR2) was digested with HindIII and SalI. Cinnamoyl-CoA reductase (CCR1) was digested with EcorI and HindIII. Phenylalanine-ammonia lyase was digested with EcoRI and SacI. 4-coumarate:CoA ligase 1 was digested with HindIII and 4-coumarate:CoA ligase 2 was digested with EcoRI and HindIII.
June 24, 2009 to June 30, 2009
Janice/Leigh:BBa_J04450 in plasmid pSB1AT3 (RFP), BBa_J04450 in plasmid pSB1AT3 (ccdB), BBa_I52001 in plasmid pSB3T5 (ccdb), BBa_J04450 in plasmid pSB3T5 (RFP) were transformed into NEB10 chemically competent cells. These are the tetracycline resistant plasmid backbones. Cultures were grown and DNA was extracted for the plasmid backbone.
July 1, 2009 to July 7, 2009
Janice/Leigh:The Arabidopsis genes were digested and were analyzed by gel electrophoresis. The double terminator, ribosome binding site, lac I promoter, pBAD promoter, and two tetracycline resistant plasmid backbones were restriction digested and ran on an agarose gel.
July 8, 2009 to July 15, 2009
Janice/Leigh:
- Performed the 3-way ligation for the lac I promoter (upstream part), ribosome binding site (RBS) (downstream part), and the destination plasmid (BBa_I52001 in pSB3C5; chloramphenicol resistance).
- Digestion: lac I promoter was digested with EcoRI and SpeI; RBS was digested with XbaI and PstI; destination plasmid was digested with EcoRI and PstI. All were digested at 37ºC for one hour and the restriction enzymes were deactivated at 80ºC for 20 minutes.
- Ligation: The lac I promoter, RBS, and the destination plasmid (BBa_I52001 in pSB3C5) were ligated and incubated for one hour.
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