Team:Colombia/Notebook

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===August 9===
===August 9===

Revision as of 17:20, 26 September 2011

Template:Https://2011.igem.org/User:Tabima

Contents

Colombia @ iGem Notebook

Here you can find our daily work in the Lab!

June

June 30:

  • Biobricks 1, 2, 3, 4 and 5 were resuspended
  • Miniprep Solutions (I, II and III) were prepared.

July

July 1

  • Biobricks 1, 2, 3, 4 and 5 were resuspended.

July 5

  • Biobrick 2 presented no colonies.
  • Colonies from bricks 1, 3, 4 and 5 were stinged.
Task:
To make LB medium (15x25mL)

July 6

  • Biobricks 1, 3 and 4 were twice plated.
  • Brick 5 didn't grow up.
  • We've added Ampiciline (3.75mL) and Kanamicine (1.875mL) to the boxed from the previous day.
  • LB medium was prepared (15x25mL).
  • Liquid LB was prepared (400mL).
Task:
To add ampiciline and tetracicline to the boxes.
Claim the liquid LB
Scrape ....
Sting biobricks 2 and 5 (no clons)
Pick up the tubes with Merceditas

July 7

  • Clons 2 and 5 didn't work out.
  • Clons 1, 3 and 4 were planted in LB liquid.
  • E. Coli inoculation in Coffee
  • Kanamicine resistence plasmid: 0.2 optic density.
  • Direct inoculation x 2 and C(-) MgCl2.
Task:
Print electroporation protocol.
Ask Juan D. Olarte about the inoculation in coffee.
Minipreps for confirmation of 1, 3 and 4.
Competent cells for clons 2 and 5.

July 8

  • Minipreps for 1, 3 and 4 were made.
  • Plant sheets of coffee into different plates: Each sheet was cut in the middle and they were incubated at 25°C and 37°C LB+Kan.
Task:
To finish the minipreps from the addition of RNAsa.
Check the E. Coli growth in coffee.

July 11

  • Minipreps have been finished. They've been planted in gel: Low concentration (25 ng/uL).
  • Transformation protocol in chemical cells.
Task:
Prepare 250 mL of SOC medium
Print the Transformation protocol for chemical cells.

July 12

  • Strains 1 and 4 have been conserved.
  • LB liquid culture was prepared again for brick 3.
  • E. Coli growth results.
Task:
Print the Transformation protocol for chemical cells.
Competent cells for clons 2 and 5.
Preserve brick 3.
Confirm bricks 1, 3 and 4 (Digestion)
Resuspend all Biobricks.
Check the E. Coli growth in coffee.

July 14

  • E. Coli didn't grow up on the sheets.
  • Chemical competent cells were made (DH5α).
  • Brick 3 was left to grow in liquid medium.
  • Bricks 2, 5, 6,7, 8, 9 and 10 were transformed and plated.
Task:
Print the Transformation protocol for chemical cells.
Preserve brick 3.
Confirm all biobricks (Digestion)
Sting the transformed bricks.
Add antibiotic to the mediums made today.

July 15

  • Brick 3 was preserved in Revco.
  • Bricks 2, 5 and 8 were stinged.
  • 25 LB+Kan boxes x 25 mL.
  • No colonies in brick 6.
  • Bricks 7, 9 and 10 were contaminated.
Task:
Print the chemical cells protocol.
Confirm all biobricks (Digestion).

July 19

  • Pass strain of Vibrio fischeri to blood agar base.
  • Check the growth of the isolates
  • Pass the transformed bricks 6, 7, 9 and 10.
  • Pass the isolates the solid media to liquid media (2,4,5 and 8)

July 21

  • Digestion to confirm No. 1, 3 and 4
Reactives 1X 6X
H2O 29,4µL 160,4 µL
Buffer N. 3 4 µL 24 µL
EcoRI 0,3 µL 1,8 µL
PstI 0,3 µL 1,8 µL
DNA 7 µL 40 µL










July 22

  • Minipreps
  • Electrophoresis of the digestions
Task:
Confirm minipreps
Re-suspend primers

July 27

  • To prepare LB and SOC medium and autoclaved
  • The bricks 7, 9 and 10 were again transformed and plated
  • Plate the brick 6.

July 30

  • Minipreps with RNase.
  • Agarose gel Electrophoresis—Results were not obtained. REPEAT!!

August

August 3

  • PCR 16S to DNA Vibrio fischeri U. Nacional
  • To expected a band of 1400 bp.
Reactives 1X 3X
H2O 6,8µL 20,4µL
Bµffer 1µL 3µL
MgCl2 0,8µL 2,4µL
dNTPs 0,2µL 0,6µL
Fw7 0,2µL 0,6µL
Rv49 0,2µL 0,6µL
Taq 0,1µL 0,3µL
DNA 1µL -
10µL















PCR Conditions

94 C for 5 min
95 C for 50 sec
55 C for 45 sec 35X
72 C for 1:30 min
72 C for 12 min
12 C forever









August 5

  • Re-suspend primers

100µM → 10 µM

Example: igem 1 → 36.1 nm → 361 µL H2O igem 2 → 32.2 nm → 322 µL H2O ➱ Vortex

Vf= 50 µL Ci= 100 µL Cf= 10 µL Vi= ?

Vi= 5 µL

  • Diluted DNA Vibrio fischeri

1244.1 ng/ µL

Ci= 1244.1 ng/ µL

Cf= 25 ng/ µL

Vf= 50 µL

Vi= 1 µL + 49 µL H2O

August 6

  • Solutions 2 and 3 of miniprep
  • Transformations of 2, 5, 7 and 13
  • Check primers


Inventory

The petri dishes with bacteria are in Q401

PCR genes (sensor, CBP and chitoporin) of Vibrio fischeri with Pfx

Name Dir Gene TM
Igem 1 Fw sensor 48,8 C
Igem 2 Rv sensor 50,8 C
Igem 3 Fw CBP 48,9 C
Igem 4 Rv CBP 49,1 C
Igem 5 Fw Chitopor 49,3 C
Igem 6 Rv Chitopor 49,4 C










August 9

  • Minipreps:
1 ✔
2 ✕
3 so-so
4 ✔
5 ✕
8 so-so
9 so-so
10 so-so
Retrieved from "http://2011.igem.org/Team:Colombia/Notebook"