Team:NYMU-Taipei/lab-protocols

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Contents

General

Transformation

ECOS(sup)TM(/sup) One-minute competent cells

  1. Prepare 42 degree Celsius water bath, plating beads and selective plates. Thaw frozen competent cells in -80 degree Celsius with room-temp. Water bath to obtain approximately 1/3 thawed state.
  2. Add DNA (DNA in 4 degree Celsius or ice-bathed plasmids or ligation mixture), whose total volume is less than 5% of volume of competent cells and vortex for 1 second. Wait 5 minutes.
  3. Incubate in 42 degree Celsius water bath for 45 seconds.
  4. Transfer onto chilled and dry selection plate media, then spread the competent cells.
  5. Immediately incubate plate at 37 degree Celsius for 12-16 hours.

High-Speed Plasmid Mini Kit Protocol

Geneaid High-Speed Plasmid Mini Kit

Add provided RNase A to the PD1 Buffer and store at 4ºC; if precipitates have formed in the PD2 Buffer, warm the buffer in a 37ºC water bath, followed by gentle shaking to dissolve. Add absolute ethanol (see the bottle label for volume) to the Wash Buffer prior to initial use.

  1. Harvesting: Transfer cultured bacterial cells to a 1.5 ml microcentrifuge tube. Centrifuge at 14-16,000 x g for 1 minute and discard the supernatant. Repeat this step if necessary.
  2. Re-suspension: Add 200 µl of PD1 Buffer (RNase A added) to the tube and re-suspend the cell pellet by vortex or pipetting.
  3. Lysis: Add 200 µl of PD2 Buffer and mix gently by inverting the tube 10 times. Do not vortex to avoid shearing the genomic DNA. Let stand at room temperature for at least 2 minutes to ensure the lysate is homologous.
  4. Neutralizatio: Add 300 µl of PD3 Buffer and mix immediately by inverting the tube 10 times. Do not vortex. Centrifuge at 14-16,000 x g for 3 minutes.
  5. DNA Binding: Place a PD Column in a 2 ml Collection Tube. Add the supernatant from Step 4 to the PD Column and centrifuge at 14-16,000 x g for 30 seconds. Discard the flow-through and place the PD Column back in the 2 ml Collection Tube.
  6. Wash: Add 400 µl of W1 Buffer into the PD Column. Centrifuge at 14-16,000 x g for 30 seconds. Discard the flow-through and place the PD Column back in the 2 ml Collection Tube. Add 600 µl of Wash Buffer (ethanol added) into the PD Column. Centrifuge at 14-16,000 x g for 30 seconds. Discard the flow through and place the PD Column back in the 2 ml Collection Tube. Centrifuge at 14-16,000 x g again for 3 minutes to dry the column matrix.
  7. DNA Elution: Transfer the dried PD Column to a new microcentrifuge tube. Add 50 µl of Elution Buffer or TE into the center of the column matrix. Let stand for at least 2 minutes to allow the Elution Buffer or TE to be completely absorbed. Centrifuge at 14-16,000 x g for 2 minutes to elute the DNA.

RE Digestion

-15μl DNA

-0.5μl E1

-0.5μl E2

-2μl 10X buffer

-2μl H2O

-20 μl total

Gel Extraction

Geneaid Gel/PCR DNA Fragments Extraction Kit

Add absolute ethanol (see the bottle label for volume) to the Wash Buffer prior to initial use.

  1. Gel Dissociation: Excise the agarose gel slice containing relevant DNA fragments and remove any extra agarose to minimize the size of the gel slice (TAE buffer is recommended for gel formation). Transfer up to 300 mg of the gel slice to a 1.5 ml microcentrifuge tube. Add 500 µl of DF Buffer to the sample and mix by vortex. Incubate at 55-60ºC for 10-15 minutes to ensure the gel slice has been completely dissolved. During incubation, invert the tube every 2-3 minutes. Cool the dissolved sample mixture to room temperature.
  2. DNA Binding: Place the DF Column in a 2 ml Collection Tube. Transfer 800 µl of the sample mixture from the previous step to the DF Column. Centrifuge at 14-16,000 x g for 30 seconds. Discard the flow-through and place the DF Column back in the 2 ml Collection Tube (If the sample mixture is more than 800 µl, repeat the DNA Binding Step).
  3. Wash: Add 400 µl of W1 Buffer into the DF Column. Centrifuge at 14-16,000 x g for 30 seconds and then discard the flow-through. Place the DF Column back in the 2 ml Collection Tube. Add 600 µl of Wash Buffer (ethanol added) into the DF Column and let stand for 1 minute. Centrifuge at 14-16,000 x g for 30 seconds and then discard the flow-through. Place the DF Column back in the 2 ml Collection Tube. Centrifuge at 14-16,000 x g for 3 minutes to dry the column matrix.
  4. DNA Elution: Transfer the dried DF Column to a new 1.5 ml microcentrifuge tube.

Add 20-50 µl of Elution Buffer or TE into the center of the column matrix. Let stand for at least 2 minutes to ensure the Elution Buffer is absorbed by the matrix. Centrifuge for 2 minutes at 14-16,000 x g to elute the purified DNA

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