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Team:Nevada/Notebook/Temp2
From 2011.igem.org
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/* Wiki Hacks - START */
/* Author: Pieter van Boheemen */
/* Team: TU Delft */
/* Thanks guys - Bill Collins */
/* +1 - Douglas Watson */
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/* Wiki Hacks - START */
/* Author: Pieter van Boheemen */
/* Team: TU Delft */
/* Thanks guys - Bill Collins */
/* +1 - Douglas Watson */
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padding-bottom:0px;
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/* Wiki Hacks - END */
Temp Calender
To edit on a specific week. Click on the edit button corresponding to the week.
Contents |
Week 5 - June 27th- July 3rd
E. Coli
Pyruvate Decarboxylase & Alcohol Dehydrogenase
JC: To insert PDC/ADH genes into the σ70 constitutive promoter plasmid, 0.5ug of σ70/pSB1A3 (2190bp) was digested with SpeI and PstI and 0.5ug of PDC/ADH/pSB1C3 (5124bp) was digested with XbaI and PstI. These digestions cut out the red fluorescent protein (rfp) tag on pSB1A3 (rfp=885bp) to aid in colony selection. Digestions were run on a 1% agarose gel, and bands obtained confirmed PDC/ADH (3054bp), pSB1C3 (2070bp), σ70/pSB1A3 (2190), and rfp (885bp). Incomplete digestion of PDC/ADH/pSB1C3 was apparent for sample #8, so the sample was re-digested before ligation.
PDC/ADH-XbaI/PstI and σ70/pSB1A3-SpeI/PstI were ligated and transformed into NEB10 β cells. Single colonies from LB-Ampicillin plates were selected and tested on LB-Amp and LB-Chlor plates. No usable colonies were obtained (all were light pink and grew on both LB-Chl and LB-Amp plates).=(
Cyano
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Enzymology
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Media
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Week 6 - July 4th-10th
E. Coli
Pyruvate Decarboxylase & Alcohol Dehydrogenase
PDC/ADH was re-digested with XbaI/PstI and run on a 1% agarose gel to confirm digestion. Bands obtained confirmed PDC/ADH (3054bp), and pSB1C3 (2070bp). PDC/ADH-XbaI/PstI and σ70/pSB1A3-SpeI/PstI were re-ligated and transformed into NEB10 β cells.
Cyano
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Enzymology
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Media
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Week 7 - July 11th-17th
E. Coli
Pyruvate Decarboxylase & Alcohol Dehydrogenase
Single colonies from LB-Amp plates were selected and tested on LB-Amp and LB-Chlor plates. Liquid cultures grown in LB-Amp and minipreps and nanodrop analysis performed.
To isolate PDC/ADH genes with the σ70 constitutive promoter, 0.5ug of PDC/ADH/σ70/pSB1A3 was digested with EcoRI and PstI. Digests were run on a 1% agarose gel, and bands confirmed σ70/PDC/ADH (3089bp) and pSB1A3 (2155bp).
Cyano
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Enzymology
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Media
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Week 8 - July 18th-24th
E. Coli
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Cyano
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Enzymology
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Media
