DNAExtractionfromBlood

實驗概要

The ChargeSwitch^?  gDNA Purification Kits allow rapid and efficient purification of  genomic DNA from small volumes of human blood. After preparing the  lysates, you may purify DNA in less than 15 minutes using the  ChargeSwitch^? Technology. Depending on the kit used, samples  may be handled individually or in an automated system using a liquid  handling robot.

實驗原理

The ChargeSwitch^? Technology (CST^?)  is a novel magnetic bead-based technology that provides a switchable  surface charge dependent on the pH of the surrounding buffer to  facilitate nucleic acid purification. In low pH conditions, the CST^?  beads have a positive charge that binds the negatively charged nucleic  acid backbone (see figure). Proteins and other contaminants are not  bound and are simply washed away in an aqueous wash buffer. To elute  nucleic acids, the charge on the surface of the bead is neutralized by  raising the pH to 8.5 using a low salt elution buffer (see figure  below). Purified DNA elutes instantly into this elution buffer, and is  ready for use in downstream applications.

主要試劑

1. ChargeSwitch^?gDNA 20 μl Blood Kits: Purifies up to 600 ng of genomic DNA from 10-20 μl of human blood.

2. ChargeSwitch^? gDNA 100 μl Blood Kit: Purifies up to 3 μg of genomic DNA from 50-100 μl of human blood.

3. ChargeSwitch^? gDNA 1 ml Blood Kit: Purifies upto 20 μg of genomic DNA from 1ml of human blood.

主要設備

1. A magnetic separation rack suitable for use with 1.5 ml microcentrifuge tubes or 96-well plates

2. Sterile, 1.5 ml microcentrifuge tubes

3. 96 x 2 ml deep well plate (if processing samples in 96-well  format; Greiner, Catalog no. 780270, Abgene, Catalog no. AB-0932, or  equivalent)

4. 96 x 300 μl U-Bottomed microtiter plate (if processing samples in 96-well format; Greiner, Catalog no. 650201 or equivalent)

5. Vortex mixer

6. 20 μl, 200 μl, and 1 ml sterile, pipette tips

實驗材料

Blood

實驗步驟

Protocol - Purification of Genomic DNA from 10-20 μl Blood Samples

This  section provides guidelines and instructions to isolate genomic DNA  from 10-20 μl samples of human blood. Note that the protocol is  optimized for efficient purification of DNA from these sample volumes.
Starting Material
Use this procedure to isolate genomic DNA from human blood samples that have been treated as follows:

• Volume: 10-20 μl of human blood

• Treatment: EDTA- or citrate-treated

• Sample state: Fresh or frozen

Before Starting
Perform the following before beginning:

1. Prepare a Lysis Mix: For each sample, mix 0.5 ml of ChargeSwitch^?  Lysis Buffer (L12) and 5 μl of Proteinase K to prepare the Lysis Mix.  If you are isolating DNA from multiple samples, you may scale up the  volume of reagents used and prepare a master Lysis Mix.

2. Vortex the tube containing the ChargeSwitch^? Magnetic Beads to fully resuspend and evenly distribute the beads in the storage buffer.

3. Prepare a Purification Mix: For each sample, mix 20 μl of ChargeSwitch^? Magnetic Beads (fully resuspended; see above) and 100 μl of ChargeSwitch^?  Purification Buffer (N5) to prepare the Purification Mix. If you are  isolating DNA from multiple samples, you may scale up the volume of  reagents used and prepare a master Purification Mix.

Preparing the Lysate
Follow the procedure below to prepare a lysate from the 10-20 μl blood sample.

1. Transfer the 10-20 μl blood sample to a sterile microcentrifuge tube (or a 96 x 2 ml deep well plate).

2. Add 0.5 ml of Lysis Mix (see above) to the sample and pipet up and down gently 5 times to mix.
   Important:  Use a 1 ml pipette tip set to 450 μl to mix the sample. Make sure that  the tip is submerged, and pipet up and down gently to avoid forming  bubbles.

3. Incubate the sample at room temperature for 10 minutes or until the sample is clear with no visible lumps.

4. Proceed to Binding DNA.

Binding DNA
Follow the procedure below to bind the DNA to the ChargeSwitch^? Magnetic Beads.

1. Gently pipet up and down the Purification Mix containing the ChargeSwitch^? Magnetic Beads to fully resuspend the beads.

2. Add 120 μl of ChargeSwitch^? Purification Mix to the digested sample (from Step 3, above) and pipet up and down gently 5 times to mix.
   Important: Use  a 1 ml pipette tip set to 550 μl to mix the sample. Make sure that the  tip is submerged, and pipet up and down gently to avoid forming bubbles.

3. Incubate at room temperature for 1 minute to allow the DNA to bind to the ChargeSwitch^? Magnetic Beads.

4. Place  the sample in the MagnaRack? (or 96-Well Magnetic Separator if using a  96-well deep well plate) for 1 minute or until the beads have formed a  tight pellet.

5. Without  removing the sample from the MagnaRack?, carefully remove the  supernatant and discard. Take care not to disturb the pellet of beads by  angling the pipette such that the tip is pointed away from the pellet  (see figure).
   Remove the sample containing the pelleted magnetic beads from the MagnaRack?. There should be no supernatant in the tube.

6. Add 500 μl of ChargeSwitch^?  Lysis Buffer (L12; without Proteinase K) to the tube and pipet up and  down gently 3 times to mix. Use a 1 ml pipette tip set to 450 μl.

7. Add 50 μl of ChargeSwitch^? Purification Buffer (N5) and pipet up and down gently 3 times to mix. Use a 1 ml pipette tip set to 500 μl.

8. Incubate at room temperature for 1 minute.

9. Place  the sample in the MagnaRack? (or 96-Well Magnetic Separator if  appropriate) for 1 minute or until the beads have formed a tight pellet.

10. Without  removing the sample from the MagnaRack?, carefully remove the  supernatant and discard. Take care not to disturb the pellet of beads by  angling the pipette such that the tip is pointed away from the pellet.

11. Proceed immediately to Washing DNA, below.

Washing DNA

1. Remove  the sample containing the pelleted magnetic beads from the MagnaRack?  (Step 11, above). There should be no supernatant in the tube.

2. Add 500 μl of ChargeSwitch^? Wash Buffer (W12) to the sample and pipet up and down gently twice to resuspend the magnetic beads.
   Important: Use  a 1 ml pipette tip set to 900 μl to mix the sample. Make sure that the  tip is submerged, and pipet up and down gently to avoid forming bubbles.

3. Place the sample in the MagnaRack? for 1 minute or until the beads have formed a tight pellet.

4. Without  removing the sample from the MagnaRack?, carefully remove the  supernatant and discard. Take care not to disturb the pellet of beads by  angling the pipette such that the tip is pointed away from the pellet.

5. Proceed to Eluting DNA.

Eluting DNA

1. Remove  the sample containing the pelleted magnetic beads from the MagnaRack?  (Step 4, above). There should be no supernatant in the tube.

2. Add 100 μl of ChargeSwitch^?  Elution Buffer (E5) (or TE Buffer, pH 8.5) to the sample and pipet up  and down gently 10 times to resuspend the magnetic beads.
   Important: Do not use water for elution. The DNA will not elute due to the poor buffering capacity of water.

3. Incubate at room temperature for 1 minute.

4. Place the sample in the MagnaRack? for 3 minutes or until the beads have formed a tight pellet.

5. Without  removing the tube from the MagnaRack?, carefully remove the supernatant  containing the DNA to a sterile microcentrifuge tube (or a 96 x 300 μl  U-bottomed microtiter plate). Take care not to disturb the pellet of  beads by angling the pipette such that the tip is pointed away from the  pellet.

6. Discard the used magnetic beads. Do not reuse the beads.

Storing DNA
Store the purified DNA at -20°C or use immediately for downstream analysis. Void repeatedly freezing and thawing DNA.
Quantitating DNA Yield
To quantitate the yield of your DNA, we recommend using the Quant-iT? PicoGreen^?  dsDNA Quantitation Kit (Catalog no. P7589) available from Invitrogen.  This kit contains the reagents necessary to allow sensitive and accurate  fluorescence-based detection of as little as 25 pg/ml of dsDNA using  the Quant-iT? PicoGreen^? dsDNA Quantitation Reagent.

Protocol - Purification of Genomic DNA from 50-100 μl Blood Samples

This  section provides guidelines and instructions to isolate genomic DNA  from 50-100 μl samples of human blood. Note that the protocol is  optimized for efficient purification of DNA from these sample volumes.
Starting Material
Use this procedure to isolate genomic DNA from human blood samples that have been treated as follows:

• Volume: 50-100 μl of human blood

• Treatment: EDTA- or citrate-treated

• Sample state: Fresh or frozen

Before Starting
Perform the following before beginning:

1. Prepare a Lysis Mix: For each sample, mix 1 ml of ChargeSwitch^?Lysis  Buffer (L12) and 10 μl of Proteinase K to prepare the Lysis Mix. If you  are isolating DNA from multiple samples, you may scale up the volume of  reagents used and prepare a master Lysis Mix.

2. Vortex the tube containing the ChargeSwitch^?Magnetic Beads to fully resuspend and evenly distribute the beads in the storage buffer.

3. Prepare a Purification Mix: For each sample, mix 40 μl of ChargeSwitch^?  Magnetic Beads (fully resuspended; see above) and 200 μl of f you are  isolating DNA from multiple samples, you may scale up the volume of  reagents used and prepare a master Purification Mix.

Preparing the Lysate
Follow the procedure below to prepare a lysate from the 50-100 μl blood sample.

1. Transfer the 50-100 μl blood sample to a sterile microcentrifuge tube or a 96 x 2 ml deep well plate.

2. Add 1 ml of Lysis Mix (see above) to the sample and pipet up and down gently 5 times to mix.
   Important: Use  a 1 ml pipette tip set to 900 μl to mix the sample. Make sure that the  tip is submerged, and pipet up and down gently to avoid forming bubbles.

3. Incubate the sample at room temperature for 10 minutes or until the sample is clear with no visible lumps.

4. Proceed to Binding DNA.

Binding DNA
Follow the procedure below to bind the DNA to the ChargeSwitch^?Magnetic Beads.

1. Gently pipet up and down the Purification Mix containing the ChargeSwitch^?Magnetic Beads to fully resuspend the beads.

2. Add 240 μl of ChargeSwitch^?Purification Mix to the digested sample (from Step 3, above) and pipet up and down gently 5 times to mix.
   Important: Use  a 1 ml pipette tip set to 900 μl to mix the sample. Make sure that the  tip is submerged, and pipet up and down gently to avoid forming bubbles.

3. Incubate at room temperature for 1 minute to allow the DNA to bind to the ChargeSwitch^?Magnetic Beads.

4. Place the sample in the MagnaRack? (or 96-Well Magnetic Separator) for 1 minute or until the beads have formed a tight pellet.

5. Without  removing the sample from the MagnaRack?, carefully remove the  supernatant and discard. Take care not to disturb the pellet of beads by  angling the pipette such that the tip is pointed away from the pellet  (see figure).

6. Proceed immediately to Washing DNA.

Washing DNA

1. Remove  the sample containing the pelleted magnetic beads from the MagnaRack?  (Step 5, above). There should be no supernatant in the tube.

2. Add 1 ml of ChargeSwitch^?Wash  Buffer (W12) to the sample and pipet up and down gently twice to  resuspend the magnetic beads. Use a 1 ml pipette tip set to 900 μl to  mix.

3. Place the sample in the MagnaRack? for 1 minute or until the beads have formed a tight pellet.

4. Without  removing the sample from the MagnaRack?, carefully remove the  supernatant and discard. Take care not to disturb the pellet of beads by  angling the pipette such that the tip is pointed away from the pellet.

5. Remove the sample containing the pelleted magnetic beads from the MagnaRack?. There should be no supernatant in the tube.

6. Add 1 ml of ChargeSwitch^?Lysis  Buffer (L12; without Proteinase K) to the tube and pipet up and down  gently 3 times to mix. Use a 1 ml pipette tip set to 900 μl to mix.

7. Add 50 μl of ChargeSwitch^?Purification Buffer (N5) and pipet up and down gently 3 times to mix. Use a 1 ml pipette tip set to 900 μl to mix.

8. Incubate at room temperature for 1 minute.

9. Place the sample in the MagnaRack? for 1 minute or until the beads have formed a tight pellet.

10. Without  removing the sample from the MagnaRack?, carefully remove the  supernatant and discard. Take care not to disturb the pellet of beads by  angling the pipette such that the tip is pointed away from the pellet.

11. Remove the tube containing the pelleted magnetic beads from the MagnaRack?.

12. Add 1 ml of ChargeSwitch^?Wash  Buffer (W12) to the tube and pipet up and down gently twice to  resuspend the magnetic beads. Use a 1 ml pipette tip set to 900 μl to  mix.

13. Place the sample in the MagnaRack? for 1 minute or until the beads have formed a tight pellet.

14. Without  removing the sample from the MagnaRack?, carefully remove the  supernatant and discard. Take care not to disturb the pellet of beads by  angling the pipette such that the tip is pointed away from the pellet.

15. Proceed to Eluting DNA, below.

Eluting DNA

1. Remove  the sample containing the pelleted magnetic beads from the MagnaRack?  (Step 14, above). There should be no supernatant in the tube.

2. Add 150 μl of ChargeSwitch^? Elution Buffer (E5) (or TE Buffer, pH 8.5) to the tube and pipet up and down gently 10 times to resuspend the magnetic beads.
   Important: Do not use water for elution. The DNA will not elute due to the poor buffering capacity of water.

3. Incubate at room temperature for 1 minute.

4. Place the sample in the MagnaRack? for 3 minutes or until the beads have formed a tight pellet.

5. Without  removing the tube from the MagnaRack?, carefully remove the supernatant  containing the DNA to a sterile microcentrifuge tube (or a 96 x 300 μl  U-bottomed microtiter plate). Take care not to disturb the pellet of  beads by angling the pipette such that the tip is pointed away from the  pellet.

6. Discard the used magnetic beads. Do not reuse the beads.

Storing DNA
Store the purified DNA at -20°C or use immediately for downstream analysis. Avoid repeatedly freezing and thawing DNA.
Quantitating DNA Yield
To quantitate yield of your DNA, use the Quant-iT? PicoGreen^?dsDNA Quantitation Kit (Catalog no. P7589).

Protocol - Purification of Genomic DNA from 1 ml blood

This section provides guidelines and instructions to isolate genomic DNA from 1 ml samples of human blood.
Starting Material
Use this procedure to isolate genomic DNA from:

• 1 ml of EDTA- or citrate-treated, fresh or frozen, human blood

• Buffy coats equivalent to 1 ml of white blood cells

Preparing the 1X RBC Lysis Buffer
The first time you use the kit, prepare 1X RBC Lysis Buffer:

• Mix the contents of the ChargeSwitch^? 10X RBC Lysis Buffer (L8; 25 ml) with 225 ml of sterile water to prepare 1X RBC Lysis Buffer (total volume = 250 ml).

• Use the 1X RBC Lysis Buffer (see Preparing the Lysate) or store at room temperature.

Preparing the Lysate
Follow the procedure below to prepare a lysate from the 1 ml blood sample.

1. To a 15 ml centrifuge tube, add the 1 ml blood sample and 10 ml of 1X RBC Lysis Buffer.

2. Mix by inverting 5 times, then incubate for 5 minutes at room temperature to lyse the red blood cells.

3. Centrifuge  the sample for 5 minutes at 2,000 x g. Carefully pour away the  supernatant, leaving a pellet of white blood cells (visible at the  bottom of the tube).

4. Add 1 ml of ChargeSwitch^?Wash Buffer (W12) by dispensing the liquid against the side of the tube. Take care not to disturb the white blood cell pellet.

Note: If the pellet is dislodged from the bottom of the tube, centrifuge the sample for 1 minute at 2,000 x g.

5. Carefully pour away the supernatant containing heme, leaving a pellet of white blood cells.

6. Shake the bottle of ChargeSwitch^?WBC  Lysis Buffer (L12) to mix (solution will appear cloudy). Add 0.5 ml to  the sample and mix by vortexing for 10 seconds (recommended) or  pipetting up and down 10 times.

7. Transfer all of the liquid (and any lumps) to a sterile microcentrifuge tube containing 1 ml of ChargeSwitch^?WBC Lysis Buffer (L12) and 20 μl of Proteinase K.

8. Pipet up and down twice to mix.

9. Incubate  the sample for 10-30 minutes at 60°C with occasional mixing (by  pipetting up and down, shaking, or vortexing) to lyse the white blood  cells. Do not proceed until the sample is clear with no visible lumps.

10. Pipet up and down 10 times to thoroughly mix the sample.
    Note: Use a 1 ml pipette tip and allow as much sample as possible to enter the tip before aspirating the liquid.

11. Proceed to Binding DNA.

Binding DNA
Follow the procedure below to bind the DNA to the ChargeSwitch^?Magnetic Beads.

1. Vortex the tube containing the ChargeSwitch^?Magnetic  Beads to fully resuspend and evenly distribute the beads in the storage  buffer. Make sure that all of the solution containing beads is at the  bottom of the tube.

2. Add 100 μl of ChargeSwitch^?Magnetic Beads to the digested sample (from Step 10, above), and pipet up and down gently twice to mix.

3. Add 100 μl of ChargeSwitch^?Purification Buffer (N5) to the sample, and pipet up and down gently 5 times to mix.
   Note: Adding the ChargeSwitch^?Purification Buffer (N5) lowers the pH of the sample, and optimizes the binding conditions.

4. Incubate at room temperature for 1 minute to allow the DNA to bind to the ChargeSwitch^?Magnetic Beads.

5. Place the sample in the MagnaRack? for 1 minute or until the beads have formed a tight pellet.

6. Without removing the tube from the MagnaRack?, carefully remove the supernatant and discard.

7. Proceed immediately to Washing DNA.

Washing DNA

1. Remove  the tube containing the pelleted magnetic beads from the MagnaRack?  (Step 6, above). There should be no supernatant in the tube.

2. Add 1 ml of ChargeSwitch^? Wash Buffer (W12) to the tube and pipet up and down gently 3 times to resuspend the magnetic beads.
   Important: Use  a 1 ml pipette tip set to 900 μl to mix the sample. Make sure that the  tip is submerged, and pipet up and down gently to avoid forming bubbles.

3. Place the sample in the MagnaRack? for 1 minute or until the beads have formed a tight pellet.

4. Without  removing the tube from the MagnaRack?, carefully remove the supernatant  and discard. Take care not to disturb the pellet of beads by angling  the pipette such that the tip is pointed away from the pellet.

5. Repeat Steps 1-4.

6. Proceed to Eluting DNA.

Eluting DNA

1. Remove  the tube containing the pelleted magnetic beads from the MagnaRack?  (Step 5, above). There should be no supernatant in the tube.

2. Add 300 μl of ChargeSwitch^?Elution Buffer (E5) (or TE Buffer, pH 8.5) to the tube and pipet up and down gently 10 times to resuspend the magnetic beads.
   Important: Do not use water for elution. The DNA will not elute due to the poor buffering capacity of water.

3. Incubate at room temperature for 5 minutes.
   Tip:  For maximum yield, mix the suspension of beads (by pipetting up and  down gently) half way through the incubation. Incubating the sample at  60°C may also improve yield.

4. Place the sample in the MagnaRack? for 5 minutes or until the beads have formed a tight pellet.

5. Without  removing the tube from the MagnaRack?, carefully remove the supernatant  containing the DNA to a sterile microcentrifuge tube. Take care not to  disturb the pellet of beads by angling the pipette such that the tip is  pointed away from the pellet.

Note: If the eluate containing the DNA is discolored, repeat Steps 4-5.

6. Discard the used magnetic beads. Do not reuse the beads.

Storing DNA

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

• Avoid  repeatedly freezing and thawing DNA. Store the purified DNA at 4°C for  short-term use or aliquot the DNA and store at -20°C for long-term  storage.

Quantitating DNA Yield

You  may estimate the yield of purified genomic DNA by checking the UV  absorbance at 260 nm or using one of the Quant-iT? DNA Assay Kits.

UV Absorbance

1. Measure the A260 of the solution using a spectrophoto-meter blanked against 10 mM Tris-HCl, pH 8.5.

 2. Calculate the amount of DNA using the formula:

DNA (μg) = A260 x 50 μg/(A260 x 1 ml) x dil’n factor x total sample volume (ml)
For DNA, A260 = 1 for a 50 μg/ml solution measured in a cuvette with an optical path length of 1 cm

Quant-iT? DNA Assay Kits

The  Quant-iT? DNA Assay Kits provide a rapid, sensitive, and accurate  method for dsDNA quantitation with minimal interference from RNA,  protein, ssDNA (primers), or other common contaminants that affect UV  absorbance. Each kit contains a state-of-the-art quantitation reagent,  pre-diluted standards for a standard curve, and a pre-made buffer to  allow fluorescence-based DNA quantitation.