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Week 10: August 8 to 13
August 8
WET LAB
Place: INDICASAT
Work Session #36 (morning)
Transformation of Competents Cells with pSB1A3, for ensure that our creation of competents cells work.
OBJECTIVE: Preparation of culture medium.
1. JM 109 strain used for cloning plasmids and growth.
This is a commercial strain that comes in a kit, were planted in a solid LB medium and then treated to make them competent.
OBJECTIVE: Transforming Competent Cells.
1. Proper markings were made.
2. One of the tubes were scored as negative control.
3. Place 2 of plasmids and resuspended in 50.
4. The nomenclature was as follows:
p-1 plate 1
1G orientation on the plate
5. The positive control was diluted 1 / 10 before taking him to the plate.
6. Was allowed to stand for 30 minutes on ice.
7. Thermal shock applied to facilitate the entry of the plasmid to the bacteria, it to 2.5 º C for 60 seconds.
8. Incubated for 5 min on ice and for the bacteria to recover from heat shock SOC 200 added to each tube including the negative control, were left incubating at 37 ° C with vigorous stirring (1.5) for 2 hours.
August 9
WET LAB
Place: INDICASAT
Work Session #37 (morning)
The Transformation works!!!
Preparation of 4 falcons tube with trasnformed cells (for growth).
(Objetives or Title):
OBJECTIVE: Creating Competent Cells.
1. Prepare the three buffers to be used.
B1 100 mL 100 mM MgCl2
B2 100 mL 100 mM CaCl2
100mL () () () = 11.098g (100x10-3) = 0.1006 g
100mL () () () = 9.5211g (100x10-3) = 0.1047 g
2. Prepare a stock solution for the buffer (100mL of 1M solution)
C1V1 = c2v2
V1 = 10mL 100mL to accommodate up
3. This solution was filtered using a syringe and special filter, everything worked within the extraction chamber.
4. From the stock solution of B1 were prepared 50 mL of 1M solution.
C1V1 = c2v2
V1 = 5mL to 50mL with water accommodate up sterilazed
5. To this solution were used falcon tubes and sterile water.
6. All solutions were kept on ice.
7. Centrifugation of the culture. 4000 rpm for 16 min.
8. The supernatant was removed and discarded in chlorine.
9. Add 1 mL of the pellet and resuspended MgCl2, expect 20 minutes and centrifuged again this time for 10 min.
10. For the B2 solution was prepared 50mL 85mm from its stock solution.
V1 = 4.25 mL to 50 mL volumetric flask with sterile water until
11. Add 20 ml of CaCl 2 and left incubating for 20 min.
12. Centrifugation was carried
13. We added in a 1000 ml eppendorf tube Buffer 3 which have:
• 850 L of Buffer????
• 150 uL glycerol molecular
• be kept on ice
August 10
Work Session #38 (morning)
Place: INDICASAT
Project Design Group: session of selection & analisys of the Gatech and Bristol Biobricks models and funtioning.
We started to study the possibility of use the CspA promoter to improve gene expression under cold shock stress.
Human Practice: preparation of concepts and definitions to the outreach campaign.
WET LAB
Work Session #39 (morning)
Minipred of our Transformed Cells.
(Objetives or Title):
--ESCRIBIR Y MEJORAR LAB---
Work Session #40 (afternoon)
--ESCRIBIR--
Finishing Miniprep, preparation of Plasmid for electrophoresis.
Left running the Electrophoresis.
August 11
WET LAB
Work Session #
(Objetives or Title):
--ESCRIBIR Y MEJORAR LAB--
Human Practice
Preparation of brochures for Outreach.
Preparation of the slides regarding to the presentation for Outreach.
Mindstorm about SynBio definitions & implications.
August 12
WET LAB
Work Session #
(Objetives or Title):
Human Practices
We were studying about the pros and cons of the SynBio and the safety regulations (for example the Synthetic Biology Report of the Presidential Commission for the Study of Bioethical Issues) for explain the risks and the safety to the people.
--ESCRIBIR Y MEJORAR LAB--
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