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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	✕
8	so-so
3	so-so
9	so-so
4	✔
10	so-so

@@ Line 331: / Line 331: @@
 |1
 |✔
-|-
+|-|5|✕
-|5
-|✕
-|-
 |2
 |✕

Team:Colombia/Notebook