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HOME	THE PROJECT	THE TEAM	PARTS SUBMITTED TO THE REGISTRY	MODELING	NOTEBOOK

Contents 1 Plates preparation 2 Biobricks amplification 3 Transformation 4 Inoculation 5 Miniprep 6 Digestion reaction 7 Gel preparation 8 Electrophoretic run 9 Gel extraction

Plates preparation

1. Autoclave LB medium with 2% agar.
2. Cool at 50°C to prevent agar polymerization.
3. Before preparing plates add antibiotic (Ampicillin 1000x or Kanamicin 200x).
4. Put about 20ml of medium per plate.
5. Leave it solidify and store at 4°C.

Biobricks amplification

1. Puncture a hole through the foil with a pipette tip (wash it everytime with bleach-distilled water-EOH 95%) into the well that corresponds to the Biobrick™-standard part that you want.
2. Add 5μl of TE buffer.
3. Rest for 20 minutes at 50°C

Transformation

1. Thaw the competent cells in ice (do not refreeze).
2. Dispense 100μl of cells into microfuge tubes on ice.
3. Add 0.1-0.3μg of plasmidic DNA or the respective amount of the ligation reaction.
4. Keep on ice for 30min.
5. Heat at 42°C for 60sec without agitation.
6. Keep on ice for 2min.
7. Add 0.9ml of LB medium at room temperature.
8. Incubate at 37°C for 1hr with agitation.
9. Pellet the cells and discard most of supernatant, leaving about 100μl.
10. Streak on plates containing appropriate antibiotics.
11. Incubate the plates overnight at 37°C.

Inoculation

1. Put 5 ml of LB in a falcon tube.
2. Add the appropriate antibiotic.
3. Pick one spot of cells from the colony with "ansa"
4. Put cells in solution.
5. Incubate the plates overnight (12 hours) at 37°C.
6. Pellet for 10 mins at 4400 rpm and discard most of supernatant.

Miniprep

1. Resuspend pelleted bacterial cells in 250μl Buffer P1 and tranfer to a microcentrifuge tube.
2. Add 250μl Buffer P2 and mix thoruoghly by inverting the tube 4-6 times.
3. Add 350μl Buffer N3 and mix immediately and thoruoghly by inverting the tube 4-6 times.
4. Centrifuge for 10 min at 13,000 rpm (17,900 x g) in a table-top microcentrifuge.
5. Apply the supernatant (from step 4) to the QIAprep spin column by decanting or pipetting.
6. Centrifuge for 30-60 s. Discard the flow-though.
7. Recommended: Wash the QIAprep spin column by adding 0.5 ml Buffer PB and centrifuging for 30-60 s. Discard the flow through.
8. Wash QIAprep spin column by eding 0.75ml Buffer PE and centrifuge for 30-60 s.
9. Discard the flow through and centrifuge for an additional 1 min to remove residual wash buffer.
10. To elude DNA, place the QIAprep column in a clean 1.5 ml microcewntrifuge tube. Add 30μl Buffer EB or water to the center of each QIAprep spin solumn, let stand for 1 min, and centrifuge for 1 min.

Digestion reaction

Note:

* E/S cut:
  enzyme 1 --> ECO R1 

  enzyme 2 --> SPE 

  buffer --> ECO R1 

• X/P cut:

enzyme 1 --> Xba
enzyme 2 --> Pst 1
buffer --> 3

• S/P cut:

enzyme 1 --> SPE
enzyme 2 --> Pst 1
buffer --> 2

• E/P cut:

enzyme 1 --> ECO R1
enzyme 2 --> Pst 1
buffer --> ECO R1

1. Mix :

- 0.5 μl of BSA
- 0.5μl of enzyme 1
- 0.5μl of enzyme 2
- 3μl of buffer
- 5μl of DNA
- 20.5μl of H2O(most pure)

1. Quick centrifuge.

Gel preparation

Electrophoretic run

Gel extraction

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