| Lab Protocol
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| A. DNA WORK
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| Agarose Gel Electrophoresis
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- Preparation of agarose gel
- Pour 100 mL of 1X TAE buffer into a conical flask.
- Add the agarose powder to the buffer in the amount with respect to the concentration of the agarose solution (e.g. add 1 g for preparing 1% agarose gel solution).
- Use a plastic wrap to cover the opening of the conical flask and microwave for approximately 2 minutes or until the agarose dissolves completely.
- Pour the agarose solution into another conical flask which specifies for holding ethidium bromide (EB) – containing solution.
- Add 1 – 2 ul of EB into the agarose solution and mix well.
- Pour the solution into a gel tray with a comb. Remove any bubbles formed.
- Allow the gel to solidify which takes approximately 30 minutes.
- Discard all the wastes into the EB waste box.
- Electrophoresis
- Remove the comb and place the solidified gel into the electrophoresis tank.
- Add TAE buffer to the tank when necessary.
- Add 6X loading buffer to the DNA sample in the ratio of 1:6 and mix well.
- Load the samples into the wells with care.
- Load 2-3 ul of marker to a well for reference.
- Run the electrophoresis at around 140V for about 30 minutes.
- Take the gel photo in the UV-illuminating machine.
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| DNA Extraction from Agarose Gel
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- Gel extraction
- Wear UV protection glasses before the gel extraction.
- Place the gel onto the transilluminator.
- Turn on the transilluminator and quickly cut the desired gel band.
- Place the cut band into an eppendorf tube for further processing.
- Discard all the wastes into the EB waste box.
- DNA extraction (Adopt from Qiagen)
- Weight the Eppendorf tube and determine the weight of the cut band.
- Add 3 volumes of extraction buffer to 1 volume of the cut get.
- Place the Eppendorf tube (with the cut gel and the extraction buffer) into 55oC water bath to dissolve all the agarose gel.
- After dissolving, add the mixture to the spin column with a collection tube.
- Centrifuge at 11,000 rpm for 1 minute.
- Discard flow through.
- Add 750 ul of washing buffer and centrifuge at 11,000 rpm for 1 minute.
- Discard the flow through and centrifuge again at 11,000 rpm for 1 minute.
- Place the collection tube to a new Eppendorf tube.
- Add 20 mL elution buffer directly to the centre of the membrane of the collection tube.
- Let it stand for approximately 3 minutes.
- Centrifuge at 11,000 rpm for 1 minutes and collect the flow through (i.e. product).
- Take a small portion of the DNA product for confirmation by gel electrophoresis.
Protocol adopt from http://www.qiagen.com/literature/render.aspx?id=201083
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| DNA Digestion
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- Add the following reagents, with the enzymes added at the last, into a tube.
- All steps should be carried out on ice.
- Mix well after addition of all the reagent.
- Incubate the mixture at 37oC for several hours.
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| Miniprep(Adopt from Qiagen)
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- Centrifuge the sample at 8,000 rpm for 1 minute.
- Discard the supernatant.
- Add 250 ul P1 buffer to resuspend the pellet (tap to suspend the pellet completely).
- Add 250 ul P2 buffer and mix gently by inverting the tube for several times.
- Add 350 ul N3 buffer and mix thoroughly. The solution should now turn cloudy.
- Centrifuge the solution at 13,000 rpm for 10 minutes.
- Transfer the supernatant to a spin column with a collection tube inside.
- Centrifuge at 12,500 rpm for 1 minute. Discard the flow through.
- Add 750 ul PE buffer to the collection tube and centrifuge at 12,500 rpm for 1 minute.
- Discard the flow through and centrifuge again to remove all remaining washing buffer.
- Place the collection tube into a new eppendorf tube.
- Add 50 ul elution buffer directly at the centre of the membrane of the collection tube.
- Let it stand for approximately 3 minutes.
- Centrifuge at 12,500 rpm for 1 minutes and collect the flow through (i.e. the product).
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