Team:Nevada/Notebook/June/Week2/3

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Week 2 - June 5-11
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<font size="5">Week 2 - June 5-11
 
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<p><font color="green">Assay for ethanol detection.  Simple one-step assay takes ethanol and NAD+ into acetylaldahyde and NADH with use of alcohol Deh (ADH) enzyme.  Final NADH concentration can be determined using its molar extinction coefficient and absorbance at 340.0 nm, and is proportionate to initial ethanol concetration.  Ethanol concentrations ranging from 0.05mM-0.25mM were assayed with 173U/mL ADH.
 
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Results: Final NADH concentrations were calculated to be off by a factor of ten due to unexpectedly low absorbancies.  Assay was repeated with increased ethanol concentrations (0.5-10mM), but absorbancies remained lower than expected. 
 
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Disscussion: It was determined that this assay was now sensitive enough for the range of ethanol concentrations that will need to be detected in our project, therefore we will use other means of ethanol detection using simple primary alcohol detection methods using oxidizing reagents.</font>
 
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<font size="5">Week 3- June 12-18
 
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<font size="5">Week 2 - June 5-11
 
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<p><font color="blue">Cyano</font> Ligation buffers prepared from 1000x stock, cloning workshop Wednesday of this week. Nevada Genomis Ceneter submission protocol reviewed. AGP knock out designed. Primers ordered to isolate genomic AGP DNA. ThiE knock out designed. E. Coli transformed with pUC57 plasmids containing, individually, GLF and INV genes. E. Coli plated on LB with ampicillin. First transformation was unsuccessful, procedure repeated successfully, five colonies for each gene were selected and grown in liquid media. Liquid cultures miniprepped using QIAgen kit and sequenced. Plasmids were successfully isolated.
 
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<p><font color="green">Assay for ethanol detection.  Simple one-step assay takes ethanol and NAD+ into acetylaldahyde and NADH with use of alcohol Deh (ADH) enzyme.  Final NADH concentration can be determined using its molar extinction coefficient and absorbance at 340.0 nm, and is proportionate to initial ethanol concetration.  Ethanol concentrations ranging from 0.05mM-0.25mM were assayed with 173U/mL ADH.
 
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Results: Final NADH concentrations were calculated to be off by a factor of ten due to unexpectedly low absorbancies.  Assay was repeated with increased ethanol concentrations (0.5-10mM), but absorbancies remained lower than expected. 
 
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Disscussion: It was determined that this assay was now sensitive enough for the range of ethanol concentrations that will need to be detected in our project, therefore we will use other means of ethanol detection using simple primary alcohol detection methods using oxidizing reagents.</font>
 
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<p><font color="black">Media</font>
 
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<font size="5">Week 3- June 12-18
<font size="5">Week 3- June 12-18
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Latest revision as of 09:33, 30 June 2011





                     Week 3- June 12-18
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