Introduction

The ChargeSwitch® Technology

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 below). 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.





ChargeSwitch® Magnetic Bead Specifications

Bead Binding Capacity:5-10 µg genomic DNA per mg
Bead Size:< 1 µm
Bead Concentration:25 mg/ml
Storage Buffer10 mM MES, pH 5.0, 10 mM NaCl, 0.1% Tween 20



Intended Use for the Kit

The ChargeSwitch® gDNA 1 ml Serum Kit allows rapid and efficient purification of genomic DNA from 0.2-1 ml samples of human serum. After preparing the lysates, you may purify DNA in less than 15 minutes using the ChargeSwitch® Technology.
 
The ChargeSwitch® gDNA 1 ml Serum Kit is designed to allow isolation of up to 200 ng of genomic DNA from 0.2-1 ml samples of fresh or frozen human serum with or without EDTA. The purified DNA is suitable for use in downstream applications including PCR and qRT-PCR.

System Specifications

Starting Material:0.2-1 ml of fresh or frozen human serum, with or without EDTA
Elution Volume:50 µl
DNA Yield:1-200 ng
DNA Size:50 bp-1.5 kb


Note:  DNA yield will vary from 1-200 ng according to sample source and storage method. The average DNA yield is 30 ng/ml of serum from healthy individuals.
 
Advantages

Use of the ChargeSwitch® gDNA 1 ml Serum Kit to isolate genomic DNA provides the following advantages:

  • Uses a magnetic bead-based technology to isolate genomic DNA without the need for hazardous chemicals, centrifugation, or vacuum manifolds
  • Rapid and efficient purification of genomic DNA from 0.2-1 ml of human serum in less than 15 minutes following sample preparation and lysis
  • Simple lysis of serum components with Proteinase K without the need for any mechanical lysis
  • Minimal contamination with RNA
  • The purified genomic DNA demonstrates improved downstream performance in applications including PCR and qRT-PCR
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General Information

User Supplied Materials

In addition to the reagents supplied with the kit, you will need to have the following materials on hand before beginning:

  • A magnetic separation rack suitable for use with 1.5 ml microcentrifuge tubes (see below)
  • Sterile microcentrifuge tubes
  • RNase A (optional; 5 mg/ml in 10 mM Tris-HCl, pH 8.5, 10 mM EDTA)
  • Vortex mixer
  • 20 µl, 200 µl, and 1 ml sterile, pipette tips



MagnaRack™

The MagnaRack™ available from Invitrogen (Catalog no. CS15000) is a two-piece magnetic separation rack for use in protocols with magnetic beads. The MagnaRack™ consists of a magnetic base station and a removable tube rack. The tube rack can hold up to 24 microcentrifuge tubes. The tube rack fits onto the magnetic base station in two different positions associating the row of 12 neodymium magnets with a single row of 12 tubes for simple ‘on the magnet’ and ‘off the magnet’ sample processing (see figure below). 
 




Safety Information

Follow the safety guidelines below when using the ChargeSwitch® gDNA 1 ml Serum Kit.

  • Treat all reagents supplied in the kit as potential irritants.
  • Always wear a suitable lab coat, disposable gloves, and protective goggles.
  • If a spill of the buffers occurs, clean with a suitable laboratory detergent and water. If the liquid spill contains potentially infectious agents, clean the affected area first with laboratory detergent and water, then with 1% (v/v) sodium hypochlorite or a suitable laboratory disinfectant.


Handling the ChargeSwitch® Magnetic Beads

Follow the guidelines below when handling the ChargeSwitch® Magnetic Beads.

  • Do not freeze the beads as this irreparably damages them. Store the beads at room temperature.
  • Always keep the beads in solution. Do not allow them to dry out as this renders them non-functional.
  • When using the beads, resuspend thoroughly in the storage buffer by vortexing before removal.
  • Discard beads after use. Do not reuse.



Elution Buffer

 
ChargeSwitch® Elution Buffer (E5; 10 mM Tris-HCl, pH 8.5) is supplied with the kit for eluting the DNA from the ChargeSwitch® Magnetic Beads. For best results, use Elution Buffer (E5) to elute the DNA. Alternatively, TE Buffer, pH 8.5-9.0 is acceptable. Note that the pH must be between 8.5-9.0 otherwise the DNA will not elute. Do not use water for elution.
 
The protocol recommends eluting the genomic DNA in 50 µl of ChargeSwitch® Elution Buffer (E5). You may vary the amount of ChargeSwitch®Elution Buffer (E5) used to obtain genomic DNA in the desired final concentration.
 
For best results, always use a volume of ChargeSwitch® Elution Buffer (E5) that is equal to or greater than the volume of ChargeSwitch® Magnetic Beads used in the protocol.
 
If the volume of ChargeSwitch® Elution Buffer (E5) is lower than the volume of beads used, DNA elution is incomplete. You may need to perform a second elution to recover all DNA.

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Isolating Genomic DNA

Introduction

This section provides guidelines and instructions to isolate genomic DNA from 0.2-1 ml samples of human serum.
 
Starting Material

Use this procedure to isolate genomic DNA from 0.2-1 ml samples of fresh or frozen human serum, with or without EDTA.
 
Materials Needed

Have the following materials on hand before beginning:

  • Serum sample(s) 
  • MagnaRack™ (Catalog no. CS15000) or other magnetic separation rack
  • 5 mg/ml RNase A (optional)
  • Sterile microcentrifuge tubes
  • Vortex mixer
  • Sterile pipette tips (20 µl, 200 µl, and 1 ml)


Components Supplied with the Kit

  • ChargeSwitch® Lysis Buffer (L19)
  • Proteinase K
  • ChargeSwitch® Magnetic Beads
  • ChargeSwitch® Purification Buffer (N7)
  • ChargeSwitch® Wash Buffer (W12)
  • ChargeSwitch® Elution Buffer (E5) or TE Buffer (not supplied; 10 mM Tris-HCl, 1 mM EDTA, pH 8.5)



Suggested Reagent Volumes 

Use the protocol in this manual to isolate genomic DNA from 1 ml samples of human serum. To isolate DNA from smaller sample volumes (i.e. 0.2-0.8 ml of human serum), scale down the volume of reagents used according to the table below.

 
Serum Volume
Reagent
1 ml
800 µl
600 µl
400 µl
200 µl
ChargeSwitch® Lysis Buffer (L19)
700 µl
560 µl
420 µl
280 µl
140 µl
Proteinase K
30 µl
30 µl
30 µl
30 µl
30 µl
RNase A (optional)
5 µl
5 µl
5 µl
5 µl
5 µl
ChargeSwitch® Purification Buffer (N7)
250 µl
200 µl
150 µl
100 µl
50 µl
ChargeSwitch® Magnetic Beads
30 µl
24 µl
18 µl
12 µl
6 µl
ChargeSwitch® Wash Buffer (W12)
1 ml
800 µl
600 µl
400 µl
200 µl
ChargeSwitch® Elution Buffer (E5)
50 µl
50 µl
50 µl
50 µl
50 µl



Before Starting

Before starting, prepare Lysis Mix as follows. For each 1 ml serum sample, mix 700 µl of ChargeSwitch® Lysis Buffer (L19) and 30 µl of Proteinase K to prepare the Lysis Mix. When isolating DNA from multiple samples, scale up the volume of reagents used and prepare a master Lysis Mix.
 
Preparing the Lysate

Follow the procedure below to prepare a lysate from a 1 ml serum sample. For smaller sample volumes, use the reagent amounts specified in the table above.
 
  1.   Place the 1 ml serum sample in a sterile microcentrifuge tube. Add 730 µl of Lysis Mix.
 
  2.   Optional: Add 5 µl of RNase A.
 
  3.   Pipet up and down 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. For smaller sample volumes, use an appropriate size pipette tip to ensure thorough mixing while avoiding bubble formation.
 
  4.   Incubate at room temperature for 20 minutes to lyse the sample. Alternatively, incubate on a rotating mixer.
 
  5.   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 250 µl of ChargeSwitch® Purification Buffer (N7) to the digested sample (from Step 4).  Note:  Adding the ChargeSwitch® Purification Buffer (N7) lowers the pH of the sample, and optimizes the binding conditions.
  3. Add 30 µl of ChargeSwitch® Magnetic Beads (from Step 1) 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.
  4. Incubate at room temperature for 2 minutes to allow the DNA to bind to the ChargeSwitch® Magnetic Beads.
  5. Place the sample in the MagnaRack™ for 3 minutes or until the beads have formed a tight pellet.
  6. 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 (see figure below).
     
     


     
  7. Proceed immediately to Washing DNA.

     

    Washing DNA

    1. Remove the tube containing the pelleted magnetic beads from the MagnaRack™ (Step 6, previous section). 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 5 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 2 minutes 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, previous section). There should be no supernatant in the tube.
    2. Add 50 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 2 minutes.
    4. Place the sample in the MagnaRack™ for 1 minute 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 PCR, qRT-PCR, or other appropriate downstream application. Avoid repeatedly freezing and thawing DNA.
     
    Quantitating DNA Yield
     
    The yield of genomic DNA obtained from 1 ml (or less) of human serum is insufficient to allow quantitation by measuring UV absorbance at 260 nm. To quantitate yield and verify the quality 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 detection of as little as 25 pg/ml of dsDNA using the Quant-iT™ PicoGreen® dsDNA Quantitation Reagent. Alternatively, you may perform Alu repeat analysis using qRT-PCR

     

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Troubleshooting

Refer to the table below to troubleshoot problems that you may encounter when purifying genomic DNA with the kit.

Problem
Cause
Solution
Low DNA yield
Incomplete lysis
  • Be sure to add Proteinase K during lysis.
  • Increase the length of incubation at room temperature.
  • Perform incubation on a rotating mixer.
 
Insufficient amount of ChargeSwitch® Magnetic Beads added
Vortex the tube containing the ChargeSwitch® Magnetic Beads to fully resuspend the beads in solution before adding them to your sample.
 
Pellet of beads disturbed or lost during binding or washing steps
  • Keep the sample in the MagnaRack when removing supernatant during the binding or washing steps.
  • Remove the supernatant without disturbing the pellet of beads by angling the pipette tip away from the pellet.
 
Incomplete dissociation of DNA from the ChargeSwitch® Magnetic Beads
Perform additional mixing of the suspension of beads (by pipetting up and down).
 
Incorrect elution conditions
  • After adding ChargeSwitch® Elution Buffer (E5) to the sample, pipet up and down to resuspend the magnetic beads before incubation.
  • Do not use water to elute DNA. Use Elution Buffer (E5) or TE, pH 8.5.
No DNA recovered
Lysis Mix added to sample instead of ChargeSwitch® Purification Buffer (N5)
You must add ChargeSwitch® Purification Buffer (N5) to your sample (in the presence of magnetic beads) to lower the pH, allowing DNA to bind to the beads.
 
Water used for elution
Do not use water for elution. The elution buffer must have a pH = 8.5-9.0 or the DNA will remain bound to the ChargeSwitch® Magnetic Beads. Use ChargeSwitch® Elution Buffer (E5) or TE, pH 8.5-9.0.
 
ChargeSwitch® Magnetic Beads stored or handled improperly
  • Store beads at room temperature. Do not freeze the beads as they will become irreparably damaged.
  • Make sure that the beads are in solution at all times and do not become dried. Beads that have dried out are non-functional.
DNA contains contaminants
Beads not fully resuspended during washing steps
Thoroughly resuspend the pellet of magnetic beads by pipetting up and down.
Eluate containing DNA is discolored
Magnetic pellet disturbed during elution
Repeat the elution step (Eluting DNA, Steps 4-5).
DNA is sheared or degraded
Lysate mixed too vigorously or small pipette tips used during mixing
  • Use a 1 ml pipette tip set to 900 µl to mix the sample.
  • Pipet up and down gently to mix.
 
Bubbles formed during mixing steps
Make sure that the pipette tip is submerged in the solution during mixing.
 
DNA repeatedly frozen and thawed
Aliquot DNA and store at 4° C or -20° C. Avoid repeated freezing and thawing.
 
DNA contaminated with DNases
Maintain a sterile environment while working (i.e. wear gloves and use DNase-free reagents).
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