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Team:Nevada/Notebook
From 2009.igem.org
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== July 8, 2009 to July 15, 2009 == | == July 8, 2009 to July 15, 2009 == | ||
| - | '''Janice/Leigh:''' | + | ==='''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). | * 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. | :# '''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. | ||
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*The 3-way ligation was transformed into NEB10 competent cells. | *The 3-way ligation was transformed into NEB10 competent cells. | ||
**20 μl of the 3-way ligation described above were added to 100 μl of NEB10 competent cells. | **20 μl of the 3-way ligation described above were added to 100 μl of NEB10 competent cells. | ||
| - | ** | + | **Chloramphenicol was spread onto the LB plates to a final concentration of 25 μg/ml. |
| + | **10, 100, and 160 μl of the transformation were added to 3 LB plates. | ||
| + | |||
Revision as of 01:10, 24 September 2009
Contents |
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
- BBa_B0014 in pSB1AK3 (double terminator)
- BBA_B0034 in pSB1A3 (ribosome binding site)
- BBA_R0011 in pSB1A3 (lac I promoter)
- BBa_I0500 in pSB2K3 (pBAD/Arac promoter).
- Made glycerol stocks for 1) to 4).
- Minipreps were done on the following Arabidopsis genes:
- cinnamoyl-CoA reductase (CCR2)
- cinnamoyl-CoA reductase (CCR1)
- phenylalanine-ammonia lyase
- 4-coumarate:CoA ligase 1
- 4-coumarate:CoA ligase 2
- Digestions:
- 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.
- 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 backbones described above.
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.
- The 3-way ligation was transformed into NEB10 competent cells.
- 20 μl of the 3-way ligation described above were added to 100 μl of NEB10 competent cells.
- Chloramphenicol was spread onto the LB plates to a final concentration of 25 μg/ml.
- 10, 100, and 160 μl of the transformation were added to 3 LB plates.
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