IGEM:MIT/2006/Notebook/2007-1-22
To Do
1. Submit sequencing before 10 AM- DONE
2. Make control culture- DONE
3. Get trained on the GC at 2 PM using cultures grown up last night- DONE
4. Make an LC of an indole-knockout colony to make them competent tomorrow- DONE
5. Make plates- DONE
6. Grow LCs of the experimental J45170.J45320s- DONE and smell them throughout the day- DONE (they smelled to both me and Veena while they were growing up)
7. Update google doc- DONE
8. Set up a smell test of J45170 with precursor added- DONE
GCMS To Do
- (VV) dilute methyl salicylate to run
- (VV) find pcnb standard concentration we used last time
- (SP) find out what we could extract isoamyl acetate into- DONE (I think; I emailed Jason to see if the procedure described at http://www.osha.gov/dts/sltc/methods/partial/pv2142/pv2142.html is viable given the GC in the lab), and then dilute isoamyl acetate to run
Another Procedure here from Leclercq-Perlat, et al:
The analysis of volatile compound composition was performed as described by Leclercq-Perlat et al. (2003b). The volatile compounds were extracted with a dynamic headspace analyzer (purge and trap concentrator, model 7695A, Hewlett-Packard, Avondale, PA). Each sample tube was connected to the apparatus and heated at 60°C for 10 min. Then it was purged with helium (99% of purity) at a flow rate of 30 mL/min for 15 min. The volatile compounds were extracted by adsorption onto a porous-polymer-adsorbent Tenax trap column (Teckmar Inc., Cincinnati, OH) at room temperature, which was then heated to 225°C for 2 min to desorb them. The compounds eluted were directly transferred at 150°C to the head of a capillary column where they were cryofocused at –150°C. Water was removed by condensation with the in-line system MCS (Hewlett-Packard) between the Tenax trap and the cryofocusing system.
The condensed compounds were separated by GC (model 6890, Hewlett-Packard) by heating the interface to 180°C for 1 min. They were automatically injected (splitless) into a nonpolar capillary column (HP-5MS; 30m x 0.25 mm and film thickness 0.25µm) at a helium flow rate of 1.6 mL/min. The oven temperature was kept at 4°C for 8 min. Then, temperature increases were programmed, from 5 to 50°C at 3°C/min, from 50 to 100°C at 5°C/min, and to the final temperature of 150°C at 10°C/min. The oven stayed at this temperature for 5 min. The GC column was connected directly to a mass selective detector (HP6890/5972). The specifications of the method used were summarized by Berger et al. (1999). The electron impact energy was set at 70 eV, and data were collected in the range of 29 to 300 atomic mass units at a scan rate of 1.68 scans/s.
Before analysis, each cheese medium sample was diluted to 1/100 with (4°C) cold Milli-Q water (Millipore system) and its pH after dilution was 5.80 ± 0.05. Prior to headspace analysis, this procedure maintained the same conditions of volatile suspension for all the samples (Mariaca and Bosset, 1997).
This analysis was carried out on 3 independent cultureS of the same type. Each culture was analyzed in triplicate by GC/MS. The mean of the abundances obtained from each of the 3 cultures was called "mean abundance."
- (SP) order vials once we get the ordering info
