Difference between revisions of "Team:CAU China/Method"

    We choose TIANGEN company's TIANamp Bacteria DNA Kit to achieve genomic DNA extraction. Protocol as follow.

• 1. Pipet 1-5 ml bacterial culture suspension in a centrifuge tube, centrifuging for 1 min at 10,000 rpm (~11,500 × g), discard supernatant as possible.
• 2. Add 200 μl Buffer GA. Mix thoroughly by vortex.
• 3. Add 20 μl Proteinase K. Mix thoroughly by vortex.
• 4. Add 220 μl Buffer GB to the sample, vortex for 15 s, and incubate at 70°C for 10 min to yield a homogeneous solution. Briefly centrifuge the 1.5 ml centrifuge tube to remove drops from the inside of the lid.
• 5. Add 220 μl ethanol (96-100%) to the sample, and mix thoroughly by vortex for 15 s. A white precipitate may form on addition of ethanol. Briefly centrifuge the 1.5 ml centrifuge tube to remove drops from the inside of the lid.
• 6. Pipet the mixture from step 5 into the Spin Column CB3 (in a 2ml collection tube) and centrifuge at 12,000 rpm (~13,400 × g) for 30 s. Discard flow-through and place the spin column into the collection tube.
• 7. Add 500 μl Buffer GD (Ensure ethanol (96-100%) has been added) to Spin Column CB3, and centrifuge at 12,000 rpm (~13,400 × g) for 30 s, then discard the flow-through and place the spin column into the collection tube.
• 8. Add 600 μl Buffer PW (Ensure ethanol (96-100%) has been added) to Spin Column CB3, and centrifuge at 12,000 rpm (~13,400 × g) for 30 s. Discard the flow-through and place the spin column into the collection tube.
• 9. Repeat Step 8.
• 10. Centrifuge at 12,000 rpm (~13,400 × g) for 2 min to dry the membrane completely.
• 11. Place the Spin Column CB3 in a new clean 1.5 ml centrifuge tube, and pipet 50-200 μl Buffer TE or distilled water directly to the center of the membrane. Incubate at room temperature (15-25°C) for 2-5 min, and then centrifuge for 2 min at 12,000 rpm (~13,400 × g).

    We choose TIANGEN company’s HiPure Plasmid Micro Kit to achieve plasmid extraction. Details as follow.

• （1） Pellet 1-5 ml of an overnight E.coli culture by centrifuging at 10,000 × g for 1 minute. Discard the supernatant.
• （2） Completely resuspend the bacterial pellet with 250 μl Buffer P1/RNase A by vortex.
• （3） Add 250 μl of the Buffer P2. Immediately mix the contents by gentle inversion (6-8 times) until the mixture becomes clear and viscous.
• （4） Add 350 μl of the Buffer NP3. Gently invert the tube 8-10 times. Pellet the cell debris by centrifuging at 13,000 × g for 1 minute.
• （5） Insert a HiPure DNA Mini Column II into a provided microcentrifuge tube. Add supernatant to the Mini Column and centrifuge at 13,000 × g for 1 minute. Discard the flow-through liquid.
• （6） Add 500 μl of the Buffer PW1 to the column. Centrifuge at 13,000 × g for 1 minute. Discard the flow-through liquid.
• （7） Add 600 μl of the Buffer PW2 to the column. Centrifuge at 13,000 × g for 1 minute. Discard the flow-through liquid. Repeat this step once more.
• （8） Centrifuge at 13,000 × g for 2 minutes without any additional Wash Solution to remove excess ethanol.
• （9） Transfer the column to a fresh collection tube. Add 50 μl Elution Buffer to the column. After putting the tube at room temperature for 1 minute, centrifuge at 13,000 × g for 1 minute. The DNA is present in the eluate.

    We choose Axygen company’s Axyprep DNA Gel Extraction Kit to achieve gel extraction. Protocol as follow.

• 1. Excise the agarose gel slice containing the DNA fragment of interest with a clean, sharp scalpel under ultraviolet illumination. Briefly place the excised gel slice on absorbent toweling to remove residual buffer. Transfer the gel slice to a piece or plastic wrap or a weighing boat. Mince the gel into small pieces and weigh. In this application, the weight of gel is regarded as equivalent to the volume.
• 2. Add a 3x sample volume of Buffer DE-A.
• 3. Heat at 75°C until the gel is completely dissolved (typically, 6-8 minutes). IMPORTANT: Gel must be completely dissolved or the DNA fragment recovery will be reduced. IMPORTANT: Do not heat the gel for longer than 10 minutes.
• 4. Add 0.5x Buffer DE-A volume of Buffer DE-B. Please make sure the contents are a uniform yellow color before proceeding.
• 5. Place a Miniprep column into a 2 ml microfuge tube (provided). Transfer the solubilized agarose from Step 4 into the column. Centrifuge at 12,000xg for 1 minute.
• 6. Discard the filtrate from the 2 ml microfuge tube. Return the Miniprep column to the 2 ml microfuge tube and add 500 μl of Buffer W1. Centrifuge at 12,000xg for 30 seconds.
• 7. Discard the filtrate from the 2 ml microfuge tube. Return the Miniprep column to the 2 ml microfuge tube and add 700 μl of Buffer W2. Centrifuge at 12,000xg for 30 seconds.
• 8. Discard the filtrate from the 2 ml microfuge tube. Place the Miniprep column back into the 2 ml microfuge tube. Add a second 700 μl aliquot of Buffer W2 and centrifuge at 12,000xg for 1 minute.
• 9. Discard the filtrate from the 2 ml microfuge tube. Place the Miniprep column back into the 2 ml microfuge tube. Centrifuge at 12,000xg for 1 minute.
• 10. Transfer the Miniprep column into a clean 1.5 ml microfuge tube (provided). To elute the DNA, add 25-30 μl of Eluent or deionized water to the center of the membrane. Let it stand for 1 minute at room temperature. Centrifuge at 12,000xg for 1 minute. Note: Pre-warming the Eluent at 65°C will generally improve elution efficiency. Note: Deionized water can also be used to elute the DNA fragments.