实验概要

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.

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.

• Micro-Tissue  Kit: Purifies up to 5 µg of genomic DNA from 1-2 mm diameter mouse ear  clips or 3-5 mg of tissue. This sample size is suitable for genomic DNA  purification from micro-dissected or laser capture micro-dissected  samples.

• Mini-Tissue  Kit: Purifies up to 30 µg of genomic DNA from 0.5 cm mouse tail tips or  approximately 25 mg of tissue. For spleen tissue, reduce tissue size to  10 mg.

Use of the ChargeSwitch^® gDNA Tissue Kits 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 micro or mini quantities  of tissue in less than 15 minutes following sample preparation and  lysis

• Simple lysis of tissues 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, restriction enzyme digestion, and Southern  blotting.

实验原理

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 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.

实验材料

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.

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, 1.5 ml microcentrifuge tubes

• Vortex mixer

• 20 µl, 200 µl, and 1 ml sterile, pipette tips

• Water bath at 55° C

实验步骤

Isolating Genomic DNA Using the Micro Tissue Kit

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:

• 1-2 mm mouse ear clips

• 3-5 mg tissue

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:

• Tissue sample(s) (see above)

• MagnaRack™ (Catalog no. CS15000) or other magnetic separation rack

• Sterile 1.5 ml microcentrifuge tubes

• Vortex mixer

• Sterile pipette tips (20 µl, 200 µl, and 1 ml)

• Water bath

Components Supplied with the Kit

• ChargeSwitch^® Lysis Buffer (L15)

• Proteinase K

• RNase A

• ChargeSwitch^® Magnetic Beads

• ChargeSwitch^® Purification Buffer (N5)

• ChargeSwitch^® Wash Buffer (W12)

• ChargeSwitch^® Elution Buffer (E5) or TE Buffer (not supplied; 10 mM Tris-HCl, 1 mM EDTA, pH 8.5)

Before Starting

Perform the following before beginning:

• Set a water bath at 55° C.

• Prepare Lysis Mix: For each sample, mix 1 ml of ChargeSwitch^®  Lysis Buffer (L15) and 10 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.

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.

1. Place the tissue sample into a sterile microcentrifuge tube.

2. Add 1 ml of Lysis Mix (see above) to the tube. Ensure that the tissue is completely immersed in the Lysis Buffer.

3. Vortex for 10-15 seconds to mix.

4. Incubate the sample overnight at 55° C until lysis is complete.

Note:  The length of the incubation step can be shortened to 1-2 hours by  vortexing the sample at least 4 times during this period.

5. Add 5 µl of RNase A to the lysate. Pipet up and down gently 5 times or until a homogeneous solution is obtained.

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 as this may result in shearing of the DNA.

6. Incubate at room temperature for 5 minutes.

7. 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 the  beads is at the bottom of the tube.

2. Add 200 µl of ChargeSwitch^® Purification Buffer (N5) to the digested tissue sample and pipet up and down gently twice to mix.

Note: Adding the ChargeSwitch^® Purification Buffer lowers the pH of the sample, and optimizes the binding conditions.

3. Add 40 µl of ChargeSwitch^® Magnetic Beads (from Step 1) to the sample and pipet up and down gently 5 times to mix.

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. 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). Proceed immediately to Washing DNA

Washing DNA

1. Remove the tube containing the pelleted magnetic beads from the MagnaRack™. 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 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 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). 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 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  55° C may also improve yield.

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 5-6.

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 US 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.

注意事项

Safety Information

Follow the safety guidelines below when using the ChargeSwitch^® gDNA Tissue 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 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.