Team:Nevada/Notebook

From 2009.igem.org

(Difference between revisions)
(July 8, 2009 to July 15, 2009)
(July 8, 2009 to July 15, 2009)
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== July 8, 2009 to July 15, 2009 ==
== July 8, 2009 to July 15, 2009 ==
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'''Janice/Leigh:'''
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==='''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.  
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**Chlo
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**Chloramphenicol was spread onto the LB plates to a final concentration of 25 μg/ml.
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**10, 100, and 160 μl of the transformation were added to 3 LB plates.
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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
  1. BBa_B0014 in pSB1AK3 (double terminator)
  2. BBA_B0034 in pSB1A3 (ribosome binding site)
  3. BBA_R0011 in pSB1A3 (lac I promoter)
  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
  5. 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).
  1. 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.
  2. 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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