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Week 16: September 19 to 24
September 19
WET LAB
I. DIGESTION
For each Eppendorf PCR was added the following:
1. 10µL DNA (residue of mini prep)
2. 32.5 µL water ultra-pure distilled water
3. 5µL buffer (P2)
4. 0.5µL BSA
5. 1µL enzyme 1 (EcoRI)
6. 1µL enzyme 2 (Spel)
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Total 50µL
There was added the 6 components to the small tube of PCR.
Note: Make sure to mix all the components in the centrifuge
7. Two (2)eppendorf tubes were incubated in the thermocycler during 1 hour at 37°C :
a. UPSTREAM (GaTech)
b. DOWNSTREAM (Bristol)
8. Once again, tubes were incubated in the thermocycler during 20 minutes at 80°C
9. We stored at -20°C until it is used for ligation.
Transformation of Competent Cells
A.
4 petri dishes were filled with 5mL of solid LB
3 positive controls and 1 negative control.
Total: 19.36µL para 20µL de LB
'Content of controls:'
Positive Control: 50µL competent cells + 2µL BBa_K410000 is resuspended.
Negative Control: 50µL competent cells
1. In 3 eppendorf tubes was added the specified amount in positive control and were resuspended.
2. Tubes in previous step remained settled in ice during 30 minutes.
3. Afterwards, cold shock carried out, the 4 eppendorf are set in water bath for a minute. (T=42°C)
4. Right after the water bath, it remained in ice during 5 minutes.
5. Added 200µL cold SOC to each eppendorf tube, then resuspended
6. Every petri dish was sealed with parafilm and was appropriately labeled; afterwards they were put in the incubator
September 20
Scientific Comunication Activities
Tree sessions:
Session 1: Civil Engineering Faculty (morning)
Session 2: Electrical Engineering Faculty (afternoon)
Session 3: Civil Engineering Faculty (afternoon)
Presentation (by the students of iGEM UTP-Panama) about the following topics:
What's SynBio?
Applications of SynBio
What's iGEM?
What are we doing? (our project)
Future projects
WET LAB
Objetives or Title:
In the picture of the result of the electrophoresis of Biobrick BBa-K381001, neither the patron nor the plasmids could be observed, for this reason the protocol was repeated the next day.
September 21
WET LAB
Ligation of BBa-K381001 and BBa-K410000
1. Buffer 10X of reaction and the T4 ligase were taken out of the fridge and they are defrosted in an ice bucket. Also the process of defrosting could have been done by centrifugation.
2. Put 11µL water in a PCR tube of 200µL
3. Added 2µL of the sample into the tube with water
4. Added 2µL buffer 10X to the tube
5. Added 1µL T4 ligase
6. Mixed appropriately shaking up the tube
7. Incubated the reaction during 20 minutes at room temperature
8. Incubated the reaction during 20 minutes at 80°C. This incubation was done to deactivated the enzyme and increase the effectiveness of the transformation
9. Stored at -24°C in order to transform later
September 22
WET LAB
(Objetives or Title):
--ESCRIBIR Y MEJORAR LAB--
September 23
WET LAB
(Objetives or Title):
--ESCRIBIR Y MEJORAR LAB--
DNA Device Submission: Explicar
September 24
GENERAL SESSION
Afternoon: Talikng about WET LAB final activities.
Design of the "SB UTP 1.0" and "SB UTP Project 1.0"
Discussion about remake the experiments with the Bristol and Gatech Biobricks (experience).
September 25
WET LAB
Sunday extra experiments session
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