IGEM:MIT/2006/Notebook/2007-8-22

Schedule

1. Run GC samples of lysed cells- DONE (NOTHING SHOWED UP! NO!)

2. 10:55- J45120 15 hour sample extraction OD=2.06, 11:15- J45181 15 hour sample extraction OD=1.98, 11:55- J45120 16 hour sample extraction OD=2.18, 12:15- J45181 16 hour sample extraction OD=1.94, 12:55- J45120 17 hour sample extraction OD=2.12, 1:15- J45181 17 hour sample extraction OD=1.84

3. Confirm flight arrangements for conference- DONE

4. Run samples above on GC

5. 3:15- Remove J45995, J45996, R0040.E0840, B0015 cultures and dilute them 1:250 at 220 RPM at 37C for 4 hours- DONE (start 4-hour growth at 3:45)

6. GC extract cultures from last night adding internal (hexyl acetate before extraction) for J45200- and J45200+PC (just want to see if we see a peak for J45600 and J45900 and J45250+PC; 600 and 900 will be 80 mL culture, 2.5 mL heptane and 1 mL will be extracted)- DONE

• Note: As Alex suggested I will dilute the J45200 and J45250 1/5 since we got a boatload last time

• We want 50 ppm internal and external standard.

• For hexyl acetate, .87 g/mL/.0001 g/mL= 1:8700 dilution; in 20 mL, 20 mL/8700 = 2.3 uL; add 2.3 uL to cultures before extraction NOTE- accidentally added this amount to J45200-, J45200+PC: For future reference, we dilute samples by 5, so 5 * 2.3 should be added, or 11.5 uL, J45250+ does not have either hexyl or octyl acetate to see whether hexyl or oxyl acetate is responsible for coelutions

• For oxyl acetate, .856 g/mL/.0002 g/mL= 1:8560 dilution; in 1 mL, 1 mL/8560 = .1168 uL; Make a 1:10 dilution in heptane and add 1.168 uL to 1 mL sample

7. Make dilutions of isoamyl acetate in heptane- wait until later to add internal and external standards- DONE

• Calculations:

500 ppm----> .876 g/mL/.0005 g/mL= 1:1752 dilution; in 2 mL heptane, 1.142 uL isoamyl acetate

200 ppm----> 1.2 mL heptane + .8 mL 500 ppm isoamyl acetate

100 ppm----> 1 mL heptane + 1 mL 200 ppm isoamyl acetate

25 ppm----> 1.5 mL heptane + .5 mL 100 ppm isoamyl acetate

5 ppm----> .8 mL heptane + .2 mL 25 ppm isoamyl acetate

7. Prepare slides for lab meeting 5-7

8. 7:45- Dilute J45995, J45996, R0040.E0840, B0015 cultures and run them at 220 RPM at 37C for 50 mins

9. Extract 200 uL from each culture and run on the plate reader

10. Email Lily about GC- DONE

11. Make fresh LCs of J45120 and J45181 (3 of each)

12. Set up J45200/J45250 time course for tomorrow