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- do tomorrow morning

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---> add 1.168 uL *1.2 = 1.4016 uL oxyl acetate

200 ppm----> 1.2 mL heptane + .8 mL 500 ppm isoamyl acetate---> add 1.168 uL octyl acetate

100 ppm----> 1 mL heptane + 1 mL 200 ppm isoamyl acetate---> add 1.752 uL octyl acetate

25 ppm----> 1.5 mL heptane + .5 mL 100 ppm isoamyl acetate ---> add 2.102 uL octyl acetate

5 ppm----> .8 mL heptane + .2 mL 25 ppm isoamyl acetate----> add 1.168 uL octyl acetate

7. Prepare slides for lab meeting 5-7- DONE

8. 7:45- Dilute J45995, J45996, R0040.E0840, B0015 cultures and run them at 220 RPM at 37C for 50 mins- SCRAP EXPERIMENT (I7100 DID NOT GROW)

PART------OD600------DILUTION

• J459951- 1.12- 1.089 mL to 23.911 mL media

• J459952- .96- 1.271 mL to 23.729 mL media

• J459953- 1.00- 1.22 mL to 23.78 mL media

• J459961- 1.16- 1.051 mL to 23.949 mL media

• J459962- 1.22- 1 mL to 24 mL media

• J459963- 1.22- 1 mL to 24 mL media

• I71001- .00 NO!!!- 1 mL to 24 mL Just for S&G

• I71002- .52- 2.346 mL to 22.654 mL media

• I71003- .48- 2.542 mL to 22.458 mL media

• B00151- .86- 1.419 mL to 23.681 mL media

• B00152- .96- 1.271 mL to 23.729 mL media

• B00153- .96- 1.271 mL to 23.729 mL media

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- do early tomorrow morning