Team:British Columbia/Protocol

Table of Contents

• Protocols

• PROJECT

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  • Inspiration
  • Description
  • Approach
  • Hardware
  • Results
  • Parts
  • Future
• LABORATORY
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  • Experimental Design
  • Protocols
  • Notebook
  • Safety
• MODEL
• HUMAN PRACTICES

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  • Integrated Human Practices
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• TEAM

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  • Team
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DNA extraction protocol

Part I: Incubation

1. Thaw filters on ice. (15 min each side)

2. Add 100 ml lysozyme (125 mg in 1000 ml TE) and 20µL RNase A (10 µg/ml) to each filter. Reseal the top with Parafilm. Incubate in a rotating incubator at 37°C for 1 hour.

3. Add 100ml Proteinase K and 100 ml 20% SDS to each filter. Reseal using Parafilm. Incubate at 55°C for 1 – 2 hours in a rotating incubator.

4. Remove lysate from sterivex filter using a 5cc syringe into a 15 ml falcon tube. Add 1 ml lysis buffer to rinse the filter out. Pool this with the lysate in the 15 ml tube.

Part II: Extraction & Centrifugation

1. Add an equal volume (about 3ml) (add 2ml) of Phenol:Chloroform:IAA (25:24:1), pH 8.0 to the lysate tube. Vortex for 10 seconds. Spin at 2500 g for 5 minutes or until aqueous layer is clear (use the J.S.-5.3 rotor, using the 50 ml falcon tubes in which the filter was stored as an adapter). Transfer aqueous layer into a new 15 ml falcon tube.

2. Add an equal volume (approx 3mL) (add 2ml) of Chloroform:IAA (24:1) to the tube containing the aqueous layer. Vortex for 10 seconds. Spin at 2500 g for 5 minutes or until aqueous layer is clear with no debris. Transfer aqueous layer into a new, labeled falcon tube. Add 1ml of TE buffer (pH 8.0) to this tube.

3. Transfer contents of the falcon tube for step 6) to an Amicon Ultra centrifuge tube. Spin at 3500 g (4000 rpm) for 10 minutes. Check to make sure there is less than 1ml liquid left in Amicon at the end of this (if not, fill up with TE and spin again). Remove filtrate to another falcon tube and save in fridge until DNA has been recovered on gel.

4. (Repeat 4) twice more (total of 3 washes with 2 ml TE). For the last wash, spin until 200 – 500 ml (pref 500) remain in Amicon. Note the final volume and transfer to a labeled 1.5 ml eppendorf tube.

5. Add 2 ml TE buffer to Amicon and spin at 3500 g for 6 minutes. Remove filtrate.

6. Add 2 ml TE buffer to Amicon and spin at 3500 g for 6 minutes. Remove filtrate.

DNA Library Ligation

We used Epicentre’s Fast-link DNA ligation kit according to their instructions here

DNA End-Fixing

We used Epicentre’s End-it DNA-repair kit according to their instructions here

Dephosphorylation of 5´-ends of DNA

We used NEB’s rSAP protocol according to their instructions here

Transformation (Chemical and Electroporation)

• Chemical transformation: We followed iGEM’s chemical transformation protocol according to their instructions here
• Electroporation: We followed NEB’s electroporation protocol fo DH5a and 10-Beta according to their instructions here

Restriction Digest

We followed NEB’s restriction digest protocols for NruI-HF, EcorI-HF, PstI-HF, SpeI-HF, and XbaI according to their instructions

Ligation (assembly of vector plasmid)

We used NEB’s T4 ligation protocol according to the instructions here

Miniprep

We used ABM’s Column-Pure Mini-prep Kit according to their instructions here

Column Purification

We used ABM’s Column-Pure PCR Cleanup Kit according to their instructions here

Making Chemically Competent Cells

1. Make a bacterial overnight culture in LB, in a 10 mL glass tube

2. The next day, add 1 mL overnight culture into 50 mL LB in a 250 mL erlenmeyer flask

3. grow to 0.5 - 0.6 OD600

4. Move cells into 50 mL falcon tube and centrifuge at 4000g for 10 min

5. Pour out the LB and resuspend in 30 ml ice-cold CaCl2

6. Centrifuge for 40 min at 4000g at 4C

7. Discard supernatant and repeat

8. Resuspend 3 mL ice-cold CaCl2 + glycerol

9. Distribute 50uL of cells into 1.5 eppendorf tubes

Gel Electrophoresis

1. Add measured out agarose to TAE (we usually use 1% gel) into an erlenmeyer flask

2. Microwave the agarose until it is completely dissolved with no particulates (can be done by running the microwave at high for 1 minute in 20 second blasts)

3. Let agarose solution cool down (cool enough to hold comfortably in your hand) and add the required amount of sybr safe

4. Fit the casting tray in the gel box (using a rubber seal) or tape the sides so that no liquid can escape

5. Swirl and pour the solution into the casting tray, add the gel comb, and allow it to solidify

6. Add enough TAE buffer into the gel box (enough to cover the gel or to the indicated line on your gel box)

7. Add your loading dye to your samples (typically 6X dye, dilute to 1X)

8. Load your gel with the samples and your DNA ladder into their respective lanes (remember to keep track of your samples)

9. Plug your gel box into your power source (with negative at the top and positive at the bottom) and run at 100V until the band is around ½ through the gel (around 1 hour)

10. Image your gel