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The ChargeSwitch® gDNA Mini and Micro Tissue Kits allow rapid and efficient purification of genomic DNA from mini (10-25 mg) or micro (3-5 mg) quantities of tissue, respectively. After preparing the lysates, you may purify DNA in less than 15 minutes using the ChargeSwitch® Technology.
Intended Use for the Kits
The ChargeSwitch® gDNA Tissue Kits are designed to allow isolation of genomic DNA from the following sources. The purified genomic DNA is suitable for use in downstream applications including PCR, restriction enzyme digestion, and Southern blotting.
Advantages
Use of the ChargeSwitch® gDNA Tissue Kits to isolate genomic DNA provides the following advantages:
System Specifications
Mini-Tissue Micro-Tissue
Starting Material: 10-25 mg 3-5 mg
Elution Volume: 250 µl 150 µl
DNA Yield: Up to 30 µg Up to 5 µg
DNA Size: > 20 kb > 20 kb
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 Buffer: 10 mM MES, pH 5.0, 10 mM NaCl, 0.1% Tween 20
Experimental Outline
Introduction
The figure below illustrates the basic steps necessary to purify genomic DNA from tissues using the ChargeSwitch® gDNA Tissue Kits.
Types of Kits
This manual is supplied with the following products.
Product | Catalog no |
ChargeSwitch® gDNA Micro Tissue Kit | CS11203 |
ChargeSwitch® gDNA Mini Tissue Kit | CS11204 |
Shipping and Storage
All components of the ChargeSwitch® gDNA Tissue Kits are shipped at room temperature. Upon receipt, store the Proteinase K and RNase A at 4° C. Store all other components at room temperature.
All components are guaranteed stable for 6 months if stored properly.
Contents
The components supplied in the ChargeSwitch® gDNA Tissue Kits are listed below. The reagents supplied are sufficient to perform 25 (Mini Tissue) or 50 (Micro Tissue) purifications.
Note: Some reagents in the kit may be provided in excess of the amount needed.
Component
|
Amount
| |
Mini Tissue | Micro Tissue | |
ChargeSwitch
® Lysis Buffer (L13)
|
50 ml
|
--
|
ChargeSwitch
® Lysis Buffer (L15)
|
--
|
50 ml
|
ChargeSwitch
® Magnetic Beads
|
3 x 1 ml
|
2 x 1 ml
|
Proteinase K (20 mg/ml in 50 mM Tris-HCl, pH 8.5, 5 mM CaCl
2, 50% glycerol)
|
500 µl
|
500 µl
|
RNase A (5 mg/ml in 10 mM Tris-HCl, pH 8.5, 10 mM EDTA)
|
250 µl
|
250 µl
|
ChargeSwitch
® Purification Buffer (N5)
|
10 ml
|
10 ml
|
ChargeSwitch
® Wash Buffer (W12)
|
50 ml
|
100 ml
|
ChargeSwitch
® Elution Buffer (E5; 10 mM Tris-HCl, pH 8.5)
|
10 ml
|
10 ml
|
User Supplied Materials
In addition to the reagents supplied with the kit, you will need to have the following materials on hand before beginning:
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). For more information, see www.invitrogen.com or call Technical Service.
Safety Information
Follow the safety guidelines below when using the ChargeSwitch® gDNA Tissue Kit.
Handling the ChargeSwitch® Magnetic Beads
Follow the guidelines below when handling the ChargeSwitch® magnetic beads.
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 150 µl (Micro Kit) or 250 µl (Mini Kit) 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.
Introduction
This section provides guidelines and instructions to isolate genomic DNA from micro quantities of tissue using the ChargeSwitch® gDNA Micro Tissue Kit (Catalog no. CS11203).
Starting Material
Use this procedure to isolate genomic DNA from:
If you wish to isolate genomic DNA from larger amounts of tissue or from mouse tail tips, see Isolating Genomic DNA Using the Mini Tissue Kit.
Materials Needed
Have the following materials on hand before beginning:
Components Supplied with the Kit
Before Starting
Perform the following before beginning:
Note: The ChargeSwitch® Lysis Buffer may appear slightly cloudy. If cloudy, shake the bottle before use until the solution becomes clear.
Preparing the Tissue Lysate
Follow the procedure below to prepare a lysate from the tissue sample.
Binding DNA
Follow the procedure below to bind the DNA to the ChargeSwitch® Magnetic Beads.
Washing DNA
Eluting DNA
Storing DNA
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
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. For more information, see www.invitrogen.com or call Technical Service
Introduction
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
|
|
Poor quality of starting material
|
Be sure to use fresh sample and process immediately after collection or freeze the sample at -80°C or in liquid nitrogen. The yield and quality of DNA isolated depends on the type and age of the starting material.
| |
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
|
| |
Low DNA yield, continued
|
Incorrect elution conditions
|
|
Incomplete dissociation of DNA from the ChargeSwitch
® Magnetic Beads
|
| |
No DNA recovered
|
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.
|
ChargeSwitch
® Magnetic Beads stored or handled improperly
|
| |
Eluate containing DNA is discolored
|
Magnetic pellet disturbed during elution
|
Repeat the elution step (
Eluting DNA).
|
RNA contamination
|
Forgot to add RNase A
|
Perform RNase A digestion prior to binding the DNA to the magnetic beads.
|
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.