Team:UTP-Panama/Week 9

WEEK 9: AUGUST 1 to 6

August 1

WET LAB

First Wet lab visit of the UTP Panama iGEM Team.
Work Session #27
Place INDICASAT
Advisor at INDICASAT: Ricardo Correa
In this day the students know all the Lab instalation, and some of the basics instruments and rules of the lab.

Work Session #27 (afternoon) Project Design and Modeling
All the team discuss the final Biobrick of our team. In this sense we select to modify and improve the Bristol 2010 Project, the Gatech 2010 Project and the UNAM-Cinvestav 2010 project. We establish the Human Practice project activities schedule.

August 2

Project Discussion & Outlines

Place INDICASAT
Work Session #28 (morning)
Preliminar Project Design of our Project with the help of our Instructor Grimaldo E. Ureña and and our advisors Zeus Capitan and Ricardo Correa, based on the selected Biobricks of the August 1 meetings and conversatory. The Wet Lab advisors establish a schedule of activities to accomplish for the students. The students organizated all the supplies and reagents to the projects first stages.

August 3

WET LAB

Place: INDICASAT (Morning)
Preparation of Buffers, organize supplies and reagents. Advisors: Ricardo, Grimaldo Elías y Lucía.

Work Session #30 (afternoon)
General Meeting, about the porject

August 4

WET LAB

Place: INDICASAT (morning)
Work Session 31
Preparation of Buffers, reagents etc, to create Competent Cells.

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 esterilazada 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

Advisor: Ricardo Correa & Grimaldo Ureña.

HUMAN PRACTICE & PROJECT DESIGN

Work Session 32 (afternoon) a). Meeting about final propousal of Human Practice Project. b). Discussion about betters ways of develop our project.

August 5

Work session #33(morning)
Preparation of more reagents for make Competents Cells. Work Session #34 (afternoon)

OBJECTIVE: Prepare buffer for Dirty Mini Prep (separation of plasmid DNA of the chromosome). 1. To prepare: Buffer P1 (kept at 4 º C) (500mL) • Tris-HCl pH 8.0 50 mM • 10 mM EDTA • RNAse A 100 mg / mL (cold) Buffer P2 (500 ml) • 200mm NaOH • SDS (1%) Buffer P3 (500 ml) • 3M potassium acetate pH5.5

2. After preparing the buffer pH were regulated to get what was needed. 3. We filter the buffer.

OBJECTIVE: Preparation of culture media. 1. Prepare culture media for the growth of competent cells. 2. Heat the solid LB. 3. Prepare 50mg/ml Ampicillin stock solution

Amp 6.25g sólido/10mL More antibiotic to 10mL water and then filtered. 4. We use a portion of the agar. 5. To this we add a volume agar ampicillin with a lower concentration, so from our stock solution:

C1V1 = c2v2

= V1

V1 = 0.02mL

6. The previous amount was changed as insufficient. Finally added to the plates 80 of ampicillin solution. 7. These dishes were left ready for the next experience.

Discussion of Human Practice project. We propouse some diferents projects as develop of a model or program to better scientific comunication of SynBio.

August 6

General Session

Work Session #35 (morning) Project Design, Conversatory about ways to implementate a model for our project.