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From 2013.igem.org

2013.igem.org

Methods

Plasmid Extraction:

1. Pellet 0.5-5 mL overnight culture.  Discard the supernatant.
2. Add 200 uL of P1 Buffer (red). Resuspend the pellet.  Transfer to a 2.0 mL tube.
3. Add 200 uL of P2 Buffer (green). Gently mix.  Incubate at room temperature for 1-3 minutes.
4. Add 400 uL of P3 Buffer (yellow). Mix thoroughly.
5. Centrifuge at 16,000g for 10 minutes.
6. Load the supernatant into a column.
7. Centrifuge for 30 seconds.
8. Discard the flow through.
9. Add 200 uL of Endo-wash buffer.  Centrifuge for 15 seconds.
10. Add 400 uL of Plasmid wash buffer.  Centrifuge 30 seconds.
11. Centrifuge for 1 minute to dry the column.
12. Place the column in a 1.5 mL tube.  Add 50 uL of sterile (autoclaved) DI water.  Let stand for 5 minutes.
13. Centrifuge for 15 seconds.

Gel Electrophoresis:

1. Add 0.6g Agarose to 75 mL of TAE buffer (1% gel)
2. Microwave for 1:15 minutes.
3. Add 6 uL ethidium bromide
4. Load the gel with a ladder and samples
5. Run for 60 minutes at 125V

Gel Purification:

1. Add 3 volumes of ADB to each volume of gel.
2. Incubate at 57 C until fully “dissolved” but not above 60 C.
3. Load dissolved solution into column.
4. Centrifuge for 30 seconds at 16,000g.  Discard the flow through.
5. Add 200 uL of DNA wash buffer to the column.  Centrifuge for 30 seconds.
6. Repeat the wash step.
7. Place the column in a new 1.5 mL tube.  Add 10 uL of elution buffer or sterilized DI water.
8. Let stand for 5 minutes.
9. Centrifuge for 30 seconds.

Transform TOP10 Cells:

1. Add 10 uL of plasmid to 50 mL of TOP10 cells.
2. Hold on ice for 15-30 minutes.
3. Heat shock for 60 seconds at 42 C.
4. Add 1000 uL of SOC with no resistance.
5. Incubate at 37 C for 2 hours.

Plate Cells:

1. Use two plates with the appropriate resistance for each sample.
2. Add 15-20 sterile glass beads to each plate.
3. Pipet 75 uL of culture onto the beads.
4. Cover the plate and roll the beads around the surface.
5. Centrifuge the remaining 925 uL of culture at 4700 rpm for 7 minutes.
6. Decant all but 100 uL of the supernatant.
7. Resuspend the pellet.
8. Plate the concentrated culture with glass beads.
9. Incubate at 37 C.

Preparation of -80 C Stock:

1. Grow cells to mid log phase.
2. Label 2 mL cryotubes with the strain, plasmid, antibiotic resistance, initials, and the date.
3. Pipet 950 uL of 30% glycerol into the tubes.
4. Add 950 uL of mid log phase culture.
5. Mix well and store in the appropriate box.
6. Update the database.

Digestion (25 uL):

• Mix 5 units of restriction enzyme, 500 ng of DNA, 2.5 uL of 10x NEBuffer, 0.5 uL of 100x BSA,
• and fill to 25 uL with sterile DI water.
• Incubate in a 37 C water bath for at least 2 hours.

Protocol for Q5® High-Fidelity 2X Master Mix

Mix all the compone

Add the samples to the PCR Thermocycler with the following protocol:

Digestion Reaction:

Ligation Reaction:

Flow Cytometry:

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