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Team:Nevada/Notebook/Temp2
From 2011.igem.org
(→Week 6 - July 4th-10th) |
(→Week 7 - July 11th-17th) |
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=='''Week 7 - July 11th-17th'''== | =='''Week 7 - July 11th-17th'''== | ||
| - | <font color=red>E. Coli <br> | + | <font color=red>E. Coli <br><br><u>Pyruvate Decarboxylase & Alcohol Dehydrogenase</u><br><br> |
| - | + | 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. | |
| + | <br><br> | ||
| + | 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). | ||
| + | <br> | ||
</font> <br> | </font> <br> | ||
<font color=blue> | <font color=blue> | ||
Revision as of 01:59, 15 July 2011
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Temp Calender
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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
Comment Here
Enzymology
Comment Here
Media
Comment Here
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
Comment Here
Enzymology
Comment Here
Media
Comment Here
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
Comment Here
Enzymology
Comment Here
Media
Comment Here
Week 8 - July 18th-24th
E. Coli
Comment Here
Cyano
Comment Here
Enzymology
Comment Here
Media
