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Genomic DNA Extraction

互联网2013-11-13

797

实验原理

The GeneCatcher™ Technology

The GeneCatcher™ Technology is a novel magnetic bead-based technology that is designed to work on a wide range of blood samples including archived or poorly stored blood samples to facilitate genomic DNA purification.

See figure below for details.

Step 1–DNA Capture–Cells are lysed and crude DNA is captured on magnetic beads leaving most of the cell debris and protein behind in solution.

Step 2–DNA Purification–Any residual protein is digested using the Protease and then washed away to leave pure intact DNA.

Step 3–Elution–The pure DNA is then eluted into a small volume ready for use in any downstream applications.

实验试剂

实验设备

24-well Magnetic Separator

100% Isopropanol

50% (v/v) Isopropanol

24-well U-bottom deep-well plates (Whatman 24-well Round Bottom UniPlate, cat. no. 7701-5102) and appropriate lids

Adjustable pipettes and aerosol barrier pipette tips

Water bath or heat block set at 65°C

实验材料

Blood sample (0.3-1 ml)

实验步骤

1) Vortex the tube containing the GeneCatcher™ Magnetic Beads to fully resuspend and evenly distribute the beads in the storage buffer.

2) Add 60 µl resuspended GeneCatcher™ Magnetic Beads to the wells of a 24-well plate.

3) Add 2.5 ml Lysis Buffer (L13) to the wells and mix the beads by gentle agitation of the plate using a plate shaker set to low speeds (800-1,000 rpm) or by gentle swirling.

4) Add 0.3-1 ml well mixed blood samples to the wells containing the beads and gently agitate the plate by swirling or placing the plate on a shaker set to low speeds.

5) Incubate at room temperature for 5 minutes to allow the DNA to bind to the beads. During incubation, agitate the samples occasionally by gently swirling the plate.

6) Place the sample on the 24-well magnetic Separator for 3 minutes. Note: You may leave the plate on the magnetic separator for up to 10 minutes to provide a convenient protocol break.

7) Without removing the plate from the Magnetic Separator, carefully remove and discard the supernatant using a 1 ml pipette without disturbing the pellet of beads by angling the pipette such that the tip is pointed away from the pellet.

8) Remove the plate from the Magnetic Separator.

9) Add 2.5 ml Lysis Buffer (L13) to the sample containing wells and gently agitate the plate by swirling or placing the plate on a shaker set to low speeds.

10) Place the sample on the 24-well magnetic Separator for 1 minute.

11) Without removing the plate from the Magnetic Separator, carefully remove and discard the supernatant using a 1 ml pipette without disturbing the pellet of beads. It is acceptable that small amount of supernatant may remain in plate at this stage if the pellet begins to dislodge.

12) Proceed immediately to Protease Digestion.

2. Protease Digestion

1) Set a water bath to 65°C.

2) Remove the plate containing the pelleted magnetic beads from the Magnetic Separator (Step 11, above).

3) Add 0.5 ml Protease Buffer and 10 µl Protease to the samples and gently agitate the plate by swirling or placing the plate on a shaker set to low speeds until the magnetic bead pellet is completely dispersed. Note: If the magnetic bead pellet does not easily disperse, warm the plate at 65°C for 5 minutes and pipet up and down gently using a 1 ml pipette tip set to 400 µl to disperse the pellet without forming any bubbles.

4) Incubate the plate at 65°C for 10 minutes and then cool the plate to room temperature (~10-20 minutes).

5) Agitate the samples by gently swirling the plate to resuspend any settled beads.

6) Proceed immediately to Washing DNA.

1) Place the sample on the Magnetic Separator for 30 seconds to 1 minute.

2) Without removing the plate from the Magnetic Separator, carefully remove and discard the supernatant using a 1 ml pipette without disturbing the pellet of beads by angling the pipette such that the tip is pointed away from the pellet.

3) Remove the plate from the Magnetic Separator.

4) Add 1 ml 50% (v/v) isopropanol to the samples and gently agitate the plate by swirling or placing the plate on a shaker set to low speeds for 15 seconds.

5) Place the sample on the Magnetic Separator for 30 seconds.

6) Without removing the plate from the Magnetic Separator, carefully remove and discard the supernatant using a 1 ml pipette without disturbing the pellet of beads.

7) Without removing the plate from the Magnetic Separator, add 150 µl Wash Buffer (W12) to the sides of the sample wells opposite to the bead pellet to ensure the bead pellet is not dislodged.

8) Incubate for 30 seconds at room temperature.

9) Without removing the plate from the Magnetic Separator, carefully remove and discard the supernatant using a 1 ml pipette without disturbing the pellet of beads.

10) Repeat Steps 8-10.

11) Proceed immediately to Eluting DNA.

1) Set a water bath to 65°C.

2) Remove the plate containing the pelleted magnetic beads (Step 11, above) from the Magnetic Separator.

3) Add 250 µl Elution Buffer (E5; 10 mM Tris-HCl, pH 8.5) to the samples and gently agitate the plate by swirling or placing the plate on a shaker set to low speeds to dislodge the pellet.

4) Incubate at 65°C for 30 minutes. For high DNA content, incubate the samples for an additional 30 minutes.

5) Remove the plate from the water bath and gently agitate the plate by swirling or placing the plate on a shaker set to low speeds until the bead pellet is completely dispersed. Note: If the pellet is not completely dispersed tip-mix without forming bubbles. For samples with high DNA content, incubate the tube over night and tip-mix gently before proceeding to the next step. Tip: If the Elution Buffer is too viscous, add additional 150-250 µl Elution Buffer (E5) and keep the samples overnight at room temperature at this stage to increase the final yield.

6) Place the sample on the Magnetic Separator until the supernatant is clear and colorless (usually 15 minutes to 1 hour, viscous samples require longer time).

7) Without removing the plate from the Magnetic Separator, carefully remove the supernatant containing the DNA using a 1 ml pipette without disturbing the pellet of beads and transfer the supernatant to a sterile tube. Note: If the supernatant is discolored, repeat Steps 6-7.

8) Discard the used magnetic beads. Do not re-use the magnetic beads.

Store the purified DNA at -20°C or use DNA for the desired downstream application.

To avoid repeated freezing and thawing of DNA, store the purified DNA at 4°C for immediate use or aliquot the DNA and store at -20°C for long-term storage.

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