Team:Nevada/Notebook

From 2009.igem.org

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==='''Janice/Leigh:'''===
==='''Janice/Leigh:'''===
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*Screen of the 3-way ligation colonies
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**Six selected colonies were cultured in LB-Chloramphenicol (25μg/ml)
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**The DNA was collected using a QIAGEN miniprep kit and concentration was determined by the Nanodrop.
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**All six colonies were digested with EcoRI to linearize, expecting a fragment length of 2.7kb.
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**Digests were run on a 1% agarose gel along with uncut DNA.  Five of the six colonies resulted in the correct band length.
==='''Chris/Joey:'''===
==='''Chris/Joey:'''===

Revision as of 23:45, 30 September 2009

Home The Team The Project Parts Submitted to the Registry Modeling Notebook

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).

Chris/Joey:

Tony/Nick:

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.

Chris/Joey:

Tony/Nick:

Sheena:

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.

Chris/Joey:

Tony/Nick:

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.

Chris/Joey:

Tony/Nick:

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.
    • 80 colonies of the 3-way ligation were screened on Amp and Chloramphenicol plates, resulting with 6 colonies that grew only on Chloramphenicol.

Chris/Joey:

Tony/Nick:

July 16, 2009 to July, 23 2009

Janice/Leigh:

  • Screen of the 3-way ligation colonies
    • Six selected colonies were cultured in LB-Chloramphenicol (25μg/ml)
    • The DNA was collected using a QIAGEN miniprep kit and concentration was determined by the Nanodrop.
    • All six colonies were digested with EcoRI to linearize, expecting a fragment length of 2.7kb.
    • Digests were run on a 1% agarose gel along with uncut DNA. Five of the six colonies resulted in the correct band length.

Chris/Joey:

Tony/Nick:

July 24, 2009 to July 31, 2009

Janice/Leigh:

Chris/Joey:

Tony/Nick:

August 1, 2009 to August 8, 2009

Janice/Leigh:

Chris/Joey:

Tony/Nick:

August 9, 2009 to August 16, 2009

Janice/Leigh:

Chris/Joey:

Tony/Nick:

August 17, 2009 to August 24, 2009

Janice/Leigh:

Chris/Joey:

Tony/Nick:

August 25, 2009 to September 1, 2009

Janice/Leigh:

Chris/Joey:

Tony/Nick:

September 2, 2009 to September 9, 2009

Janice/Leigh:

Chris/Joey:

Tony/Nick:

September 10, 2009 to September 17, 2009

Janice/Leigh:

Chris/Joey:

Tony/Nick:

September 18, 2009 to September 25, 2009

Janice/Leigh:

Chris/Joey:

Tony/Nick:

September 26, 2009 to October 3, 2009

Janice/Leigh:

Chris/Joey:

Tony/Nick:

October 4, 2009 to October 11, 2009

Janice/Leigh:

Chris/Joey:

Tony/Nick:

October 12, 2009 to October 19, 2009

Janice/Leigh:

Chris/Joey:

Tony/Nick:

October 20, 2009 to October 27, 2009

Janice/Leigh:

Chris/Joey:

Tony/Nick:

October 28, 2009 to November 4, 2009

Janice/Leigh:

Chris/Joey:

Tony/Nick:

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