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Contents 1 iGemColombia Notebook 1.1 June 1.1.1 June 30: 1.2 July 1.2.1 July 1 1.2.2 July 5 1.2.3 July 6 1.2.4 July 7 1.2.5 July 8 1.2.6 July 11 1.2.7 July 12 1.2.8 July 14 1.2.9 July 15 1.2.10 July 19 1.2.11 July 21 1.2.12 July 22 1.2.13 July 27 1.2.14 July 30 1.3 August

iGemColombia 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!!

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August