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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).
Chris/Joey:
Tony/Nick:
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.
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).
- 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.
- 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:
- Screening of the 3-way ligation (lac promoter/RBS/backbone) 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.
- 3-way ligation of RBS (BBa_R0011), pBAD promoter (BBa_I0500), and Chloramphenicol backbone (pSB3C5)
- The upstream part, pBAD promoter, was digested with EcoRI and SpeI
- The downstream part, RBS, was digested with XbaI and PstI
- The Chloramphenicol bakcbone was digested with EcoRI and PstI
- All digests were incubated for one hour at 37C followed by a 20 minute deactivation step at 80C
- 4μl of each of the three digests were used in a final 20μl ligation reaction with a one hour incubation at root temperature, followed by a 20 minute deactivation step at 80C
- The ligation was transformed into NEB10 cells and volumes of 10, 50, 100μl, and the rest was spread on LB plates containing Chloramphenicol (25μg/ml)
- The plates were incubated overnight at 37C
Chris/Joey:
Tony/Nick:
July 24, 2009 to July 31, 2009
Janice/Leigh:
- Screening of the 3-way ligation (pBAD promoter/RBS/backbone) colonies
- 7 colonies were selected and cultured in 3ml of LB-Chloramphenicol (25μg/ml)
- Each colony was digested with PstI to linearize and a double digest of PstI/EcoRI to cut out the pBAD promoter(1.2kb)
- The digests were run on a 1% agarose gel, none of which resulted with the expected fragments lengths.
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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