"
Team:Austin Texas/Week of July 16 to July 22
From 2012.igem.org
Contents |
Notebook for week of July 16 to July 22
Design of Converter Plasmid
- Razan
- 7/16- Tested overlap regions using overlap PCR.
- 7/17- Tested overlap regions 2 pieces at a time using gibson. Ordered new primers to increase backbone overlap region.
Design of Reporter Plasmid
Spinach Aptamer
- Logan
- 7/16 - Ran PCR reactions on many random colonies from the plate of the construct with mCherry 3' of SpA.
- 7/17 - Colony PCR of both 3' and 5' constructs (as in 7/16). Ran GE for PCR products from 7/16 - Found many plasmid with correct insert.
- 7/18 - Started cultures at 7:30 AM, came in at 11 and still not mid log phase so continued culturing and will dilute next day. Transformed pAAV-miniCMV-mCherry plasmid into Bl-21 AI cells to use as control in plate reader exp.
- 7/19 - Diluted cultures from 7/18 to use in plate reader exp. After dilution, grew 1.5 hours, added 25 uM DFHBI(gfp-like fluorophore) and cultured further 30 mins. Induced with arabinose, and ran SpA, mCherry, and the 5' construct in 96 well plate for 3.5 hours monitoring fluorescent wavelengths of both SpA and mCherry.
- 7/20 - Result of plate reader exp: no fluorescence. Did not wait long enough for induction. Pelleted the plate reader cultures from 7/19 (grown with DFHBI and arabinose induced overnight) - Under blue UV box: mCherry - no fluoresence, SpA - little fluorescence, 5' construct - glowing red and green. Transformed 3' construct plasmid in Bl-21 AI, will sequence 7/23.
