Team:UTK-Knoxville/methods
From 2013.igem.org
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<h2><span class="mw-headline" id="Background">Methods</span></h2> | <h2><span class="mw-headline" id="Background">Methods</span></h2> | ||
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<p dir="ltr"><strong id="docs-internal-guid-3c286855-68a8-2624-ed13-2832044f6359">Plasmid Extraction:</strong></p> | <p dir="ltr"><strong id="docs-internal-guid-3c286855-68a8-2624-ed13-2832044f6359">Plasmid Extraction:</strong></p> |
Latest revision as of 16:25, 10 July 2013
Methods
Plasmid Extraction:
- Pellet 0.5-5 mL overnight culture. Discard the supernatant.
- Add 200 uL of P1 Buffer (red). Resuspend the pellet. Transfer to a 2.0 mL tube.
- Add 200 uL of P2 Buffer (green). Gently mix. Incubate at room temperature for 1-3 minutes.
- Add 400 uL of P3 Buffer (yellow). Mix thoroughly.
- Centrifuge at 16,000g for 10 minutes.
- Load the supernatant into a column.
- Centrifuge for 30 seconds.
- Discard the flow through.
- Add 200 uL of Endo-wash buffer. Centrifuge for 15 seconds.
- Add 400 uL of Plasmid wash buffer. Centrifuge 30 seconds.
- Centrifuge for 1 minute to dry the column.
- Place the column in a 1.5 mL tube. Add 50 uL of sterile (autoclaved) DI water. Let stand for 5 minutes.
- Centrifuge for 15 seconds.
Gel Electrophoresis:
- Add 0.6g Agarose to 75 mL of TAE buffer (1% gel)
- Microwave for 1:15 minutes.
- Add 6 uL ethidium bromide
- Load the gel with a ladder and samples
- Run for 60 minutes at 125V
Gel Purification:
- Add 3 volumes of ADB to each volume of gel.
- Incubate at 57 C until fully “dissolved” but not above 60 C.
- Load dissolved solution into column.
- Centrifuge for 30 seconds at 16,000g. Discard the flow through.
- Add 200 uL of DNA wash buffer to the column. Centrifuge for 30 seconds.
- Repeat the wash step.
- Place the column in a new 1.5 mL tube. Add 10 uL of elution buffer or sterilized DI water.
- Let stand for 5 minutes.
- Centrifuge for 30 seconds.
Transform TOP10 Cells:
- Add 10 uL of plasmid to 50 mL of TOP10 cells.
- Hold on ice for 15-30 minutes.
- Heat shock for 60 seconds at 42 C.
- Add 1000 uL of SOC with no resistance.
- Incubate at 37 C for 2 hours.
Plate Cells:
- Use two plates with the appropriate resistance for each sample.
- Add 15-20 sterile glass beads to each plate.
- Pipet 75 uL of culture onto the beads.
- Cover the plate and roll the beads around the surface.
- Centrifuge the remaining 925 uL of culture at 4700 rpm for 7 minutes.
- Decant all but 100 uL of the supernatant.
- Resuspend the pellet.
- Plate the concentrated culture with glass beads.
- Incubate at 37 C.
Preparation of -80 C Stock:
- Grow cells to mid log phase.
- Label 2 mL cryotubes with the strain, plasmid, antibiotic resistance, initials, and the date.
- Pipet 950 uL of 30% glycerol into the tubes.
- Add 950 uL of mid log phase culture.
- Mix well and store in the appropriate box.
- 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
Component |
25 µl Reaction |
50 µl Reaction |
Final Concentration |
Q5 High-Fidelity 2X Master Mix |
12.5 µl |
25 µl |
1X |
10 µM Forward Primer |
1.25 µl |
2.5 µl |
0.5 µM |
10 µM Reverse Primer |
1.25 µl |
2.5 µl |
0.5 µM |
Template DNA |
variable |
variable |
< 1,000 ng |
Nuclease-Free Water |
to 25 µl |
to 50 µl |
|
Add the samples to the PCR Thermocycler with the following protocol:
STEP |
TEMP |
TIME |
Initial Denaturation |
98°C |
30 seconds |
25–35 Cycles |
98°C |
5–10 seconds |
*50–72°C |
10–30 seconds |
|
72°C |
20–30 seconds/kb |
|
Final Extension |
72°C |
2 minutes |
Hold |
4–10°C |
|
Digestion Reaction:
Ligation Reaction:
Flow Cytometry:
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