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.
|Bead Binding Capacity:||5-10 µg genomic DNA per mg|
|Bead Size:||< 1 µm|
|Bead Concentration:||25 mg/ml|
|Storage Buffer||10 mM MES, pH 5.0, 10 mM NaCl, 0.1% Tween 20|
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.
|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.
Use of the ChargeSwitch gDNA 1 ml Serum Kit to isolate genomic DNA provides the following advantages:
In addition to the reagents supplied with the kit, you will need to have the following materials on hand before beginning:
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).
Follow the safety guidelines below when using the ChargeSwitch gDNA 1 ml Serum Kit.
Follow the guidelines below when handling the ChargeSwitch Magnetic Beads.
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.
This section provides guidelines and instructions to isolate genomic DNA from 0.2-1 ml samples of human serum.
Use this procedure to isolate genomic DNA from 0.2-1 ml samples of fresh or frozen human serum, with or without EDTA.
Have the following materials on hand before beginning:
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.
ChargeSwitch Lysis Buffer (L19)
RNase A (optional)
ChargeSwitch Purification Buffer (N7)
ChargeSwitch Magnetic Beads
ChargeSwitch Wash Buffer (W12)
ChargeSwitch Elution Buffer (E5)
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.
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.
Follow the procedure below to bind the DNA to the ChargeSwitch Magnetic Beads.
Refer to the table below to troubleshoot problems that you may encounter when purifying genomic DNA with the kit.
Low DNA yield
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
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
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
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
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).
For Research Use Only. Not for use in diagnostic procedures.