IGEM:MIT/2006/Notebook/2007-8-22: Difference between revisions

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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
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
NOTE- I think that growing up the cultures in 125-mL flasks from the start altered the growth curves


3. Confirm flight arrangements for conference- DONE
3. Confirm flight arrangements for conference- DONE


4. Run samples above on GC
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
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) and external standards (octyl acetate after extraction) for J45200 and J45250 (just want to see if we see a peak for J45600 and J45900)- DONE
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
*Note: As Alex suggested I will dilute the J45200 and J45250 1/5 since we got a boatload last time
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*We want 50 ppm internal and external standard.
*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
*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
*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


3. Make dilutions of isoamyl acetate in heptane (will add external and internal standards later if we see that they do not coelute with isoamyl acetate)- DOEN
7. Make dilutions of isoamyl acetate in heptane- wait until later to add internal and external standards- DONE


*Calculations:
*Calculations:


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


200 ppm----> 1.2 mL heptane + .8 mL 500 ppm isoamyl acetate
*I71001- .00 NO!!!- 1 mL to 24 mL Just for S&G


100 ppm----> 1 mL heptane + 1 mL 200 ppm isoamyl acetate
*I71002- .52- 2.346 mL to 22.654 mL media


25 ppm----> 1.5 mL heptane + .5 mL 100 ppm isoamyl acetate
*I71003- .48- 2.542 mL to 22.458 mL media


5 ppm----> .8 mL heptane + .2 mL 25 ppm isoamyl acetate
*B00151- .86- 1.419 mL to 23.681 mL media


7. Prepare slides for lab meeting 5-7
*B00152- .96- 1.271 mL to 23.729 mL media


8. 7:00- Dilute J45995, J45996, R0040.E0840, B0015 cultures and run them at 220 RPM at 37C for 50 mins
*B00153- .96- 1.271 mL to 23.729 mL media


9. Extract 200 uL from each culture and run on the plate reader
9. Extract 200 uL from each culture and run on the plate reader
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10. Email Lily about GC- DONE
10. Email Lily about GC- DONE


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


12. Set up J45200/J45250 time course for tomorrow
12. Set up J45200/J45250 time course for tomorrow- do early tomorrow morning

Latest revision as of 16:33, 20 November 2007

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

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