Team:UTP-Panama/Week 15

Week 15: September 12 to 17

September 12

WET LAB

We grew the BBa-K381001 in liquid LB + chloramphenicol to then continue with DIRTY MINIPREP in order to extract its plasmids.

1. Took out of the fridge 2 petri dishes with transformed bacteria with BBa-K381001
2. It was used:
   1. 9.7µL of chloramphenicol
   2. and 10mL of liquid LB
3. Prepared (+) and (-) controls for each petri dish in falcon tubes
4. Each falcon tube of (+) control contains:
   1. 9.7µL of chloramphenicol/10mL of Liquid LB
   2. 5mL of liquid LB
   3. Aliquot of the cultivation
5. The negative controls had almost the same content, excluding chloramphenicol
6. The 4 falcon tubes were collocated in the incubator until next day

September 13

Scientific Comunication Activities

Presentation (by the students of iGEM UTP-Panama) in the Civil and Electrical Faculties about:
What's SynBio?
Applications of SynBio
What's iGEM?
What are we doing? (our project)
Future projects

WET LAB

Objetives: DIRTY MINI PREP BBa-K381001 part Cultivation 9/6/2011 1. Appropriately shook the tubes where the cultivations were.
2. In each eppendorf 1.5mL of cultivation (9/6/2011) control (+)
3. In each eppendorf 1.5mL of cultivation (9/6/2011) control (-)
4. Centrifuged 13000rpm, 1 min
5. Supernatant of eppendorf 6 tubes were eliminated
6. Resuspended in 300µL buffer RNasa (P1)
7. Added 300µL of buffer (P2)
8. Mixed by inversion
9. It was kept in room temperature during 5 minutes
10. Added 300µL of buffer (P3)
11. Mixed by inversion
12. Kept in ice during 5 minutes
13. Centrifuged 13000rpm, 10 min
14. Extracted the supernatant and it was deposited in new eppendorf tubes carefully, avoiding to touch the pellets.
15. Added to each tube 1000µL of PCA (Phenol:Chloroform:Isoamyl alcohol)
16. Mixed by inversion
17. Centrifuged 13000rpm, 5 min
18. Transferred 750µL aqueous phase to new tubes properly labeled.
19. Tubes with plasmids and isopropanol were centrifuged 13000rpm, 15 min
20. Then isopropanol was removed
21. Added 500µL EtOH 70% to wash it
22. Mixed by inversion
23. Again centrifuged 13000rpm, 5 min
24. Eliminated EtOH 70% and were dried (air dry technique)
25. Resuspended in 30µL of H20 ultra-pure (it was cold)
26. Tubes were stored in a white box properly labeled

Note: Volumes of 500µL EtOH 70% and 30µL of H20 ultra-pure refer to that specific amount for each tube.

September 17

GENERAL SESSION

Morning: Talking about WET LAB final activities. Objetives:
Design of Experiment Test of cell growth transformed with BBa_K328003 and BBa_K328001
1. To grow petri dishes at 37°C, 30°C, 20°C, 10°C, 0°C, -10°C
2. In different substrates
a. LB
b. Water and oil
c. Water and sugar
d. Minimum Essential Medium
e. Salt solution
All the Team (under Human Practice Direction):