Genomic DNA Extraction

The GeneCatcher™ gDNA Blood Kits allow rapid and efficient extraction of genomic DNA (gDNA) from human blood including archived or poorly stored blood samples.  Genomic DNA is extracted from blood samples using the cost-effective, user-friendly GeneCatcher™ Technology without the use of centrifugation.  The purified DNA is suitable for use in any downstream application of choice including PCR and restriction enzyme digestion.
 
Advantages

The GeneCatcher™ gDNA Blood Kits provide a flexible solution for large-scale genotyping and biobanking projects due to the following advantages:

• Scalable extraction from a wide range of blood sample volumes
• Superior performance on difficult blood samples
• High purity and yield of extracted genomic DNA
• Single well extraction to facilitate sample tracking in clinical applications
• Quick benchtop protocol

The GeneCatcher™ Technology

The GeneCatcher™ Technology is a novel magnetic bead-based technology that is designed to work on a wide range of blood samples including archived or poorly stored blood samples to facilitate genomic DNA purification.
See figure below for details.
  
Step 1–DNA Capture–Cells are lysed and crude DNA is captured on magnetic beads leaving most of the cell debris and protein behind in solution.
 
Step 2–DNA Purification–Any residual protein is digested using the Protease and then washed away to leave pure intact DNA.
 
Step 3–Elution–The pure DNA is then eluted into a small volume ready for use in any downstream applications.






System Specifications

(*The gDNA yield will depend on the sample volume and white blood cell count.)

Shipping and Storage

All components of the GeneCatcher™ gDNA (genomic DNA) Blood Kits are shipped at room temperature. Upon receipt, store components as follows:

• Store Protease at 4 °C
• Store the remaining kit components at room temperature

All components are guaranteed stable for 6 months when stored properly. 

Contents

The components supplied in the GeneCatcher™ gDNA Blood Kits are listed below.

The reagents supplied are sufficient to perform:

• 96 purifications (CS21101) using up to 1 ml blood
• 66 purifications (CS21110) using 3 ml blood
• 20 purifications (CS21110) using 10 ml blood

Since the amount of reagents used is different for various starting volumes of blood, it is normal for some reagents to be in excess in the kit after performing the specified number of purifications.

• Fresh, whole blood
• Blood collected in the presence of anti-coagulants such as EDTA, heparin, or citrate.
• Frozen blood samples or blood samples exposed to repeated freeze-thaw cycles
• Old, archived blood samples and degraded samples

• Maintain a sterile environment when handling DNA to avoid any contamination from DNases
• Ensure that no DNase is introduced into the solutions supplied with the kit
• Make sure that all equipment coming in contact with DNA is sterile, including pipette tips and tubes
• Perform the recommended wash steps during the purification to obtain the best results

• During the mixing and washing steps of the GeneCatcher™ Magnetic Beads, mix beads by gentle agitation using a plate shaker set to low speeds (800-1,000 rpm) when purifying gDNA from 0.3-1 ml blood samples or by gently inverting the tube or pipetting up and down gently without forming bubbles (tip-mix) when purifying gDNA from 3-10 ml blood samples.
• Do not allow the beads to dry as drying reduces the bead efficiency.
• To aspirate the supernatant after bead washing, place the pipette tip away from the beads by angling the pipette such that the tip is pointed away from the pellet and carefully remove the supernatant without disturbing or removing any beads.
• Do not freeze the magnetic beads, as freezing damages the beads and cannot be used for nucleic acid purifications.

• Treat all reagents supplied in the kit as potential irritants.
• Always wear a suitable lab coat, disposable gloves, and protective goggles when handling whole blood samples.
• Dispose of blood samples and washes during the purification procedure as biohazardous waste.

Introduction

Instructions for isolating gDNA from 0.3-1 ml human blood are described below. To isolate gDNA from 3-10 ml human blood.  The procedure is designed for isolating gDNA using the GeneCatcher™ Magnetic Beads procedure.
 
24-well Magnetic Separator

You will need a magnetic rack (separator) for use with the GeneCatcher™ gDNA 0.3-1 ml Blood Kit. We recommend using the 24-well Magnetic Separator (catalog No. CS15024) for 24-well plates to obtain the best results. Other magnetic separators may not provide similar magnetic strength or may not be compatible with the volumes used in the protocol.

The 24-well Magnetic Separator is a magnetic separation rack (see below) for use in protocols with magnetic beads. The 24-well plate fits onto the magnetic rack associating the row of 6 neodymium magnets with the 24 wells for simple sample processing using magnetic beads (see figure below).










Materials Needed

• Blood sample (0.3-1 ml)
• 24-well Magnetic Separator
• 100% Isopropanol
• 50% (v/v) Isopropanol
• 24-well U-bottom deep-well plates (Whatman 24-well Round Bottom UniPlate, cat. no. 7701-5102) and appropriate lids
• Adjustable pipettes and aerosol barrier pipette tips
• Water bath or heat block set at 65°C

 
Optional: Plate shaker with a minimum orbit of 2 mm operating at 800-1,000 rpm.


Components supplied with the kit

• GeneCatcher™ Lysis Buffer (L13)
• Protease
• Protease Buffer
• GeneCatcher™ Magnetic Beads
• GeneCatcher™ Wash Buffer (W12)
• GeneCatcher™ Elution Buffer (E5)

Binding DNA

Follow the procedure below to bind DNA to the GeneCatcher™ Magnetic Beads.

1. Vortex the tube containing the GeneCatcher™ Magnetic Beads to fully resuspend and evenly distribute the beads in the storage buffer.
2. Add 60 µl resuspended GeneCatcher™ Magnetic Beads to the wells of a 24-well plate.
3. Add 2.5 ml Lysis Buffer (L13) to the wells and mix the beads by gentle agitation of the plate using a plate shaker set to low speeds (800-1,000 rpm) or by gentle swirling.
4. Add 0.3-1 ml well mixed blood samples to the wells containing the beads and gently agitate the plate by swirling or placing the plate on a shaker set to low speeds.
5. Incubate at room temperature for 5 minutes to allow the DNA to bind to the beads. During incubation, agitate the samples occasionally by gently swirling the plate.
6. Place the sample on the 24-well magnetic Separator for 3 minutes. Note: You may leave the plate on the magnetic separator for up to 10 minutes to provide a convenient protocol break.
7. Without removing the plate from the Magnetic Separator, carefully remove and discard the supernatant using a 1 ml pipette without disturbing the pellet of beads by angling the pipette such that the tip is pointed away from the pellet.
8. Remove the plate from the Magnetic Separator.
9. Add 2.5 ml Lysis Buffer (L13) to the sample containing wells and gently agitate the plate by swirling or placing the plate on a shaker set to low speeds.
10. Place the sample on the 24-well magnetic Separator for 1 minute.
11. Without removing the plate from the Magnetic Separator, carefully remove and discard the supernatant using a 1 ml pipette without disturbing the pellet of beads. It is acceptable that small amount of supernatant may remain in plate at this stage if the pellet begins to dislodge.
12. Proceed immediately to Protease Digestion.

Protease Digestion

1. Set a water bath to 65°C.
2. Remove the plate containing the pelleted magnetic beads from the Magnetic Separator (Step 11, above).
3. Add 0.5 ml Protease Buffer and 10 µl Protease to the samples and gently agitate the plate by swirling or placing the plate on a shaker set to low speeds until the magnetic bead pellet is completely dispersed. Note: If the magnetic bead pellet does not easily disperse, warm the plate at 65°C for 5 minutes and pipet up and down gently using a 1 ml pipette tip set to 400 µl to disperse the pellet without forming any bubbles.
   
     
    
4. Incubate the plate at 65°C for 10 minutes and then cool the plate to room temperature (~10-20 minutes).
5. Agitate the samples by gently swirling the plate to resuspend any settled beads.
6. Proceed immediately to Washing DNA.

 
Washing DNA

Add 0.5 ml 100% isopropanol to the samples and gently agitate the plate by swirling or placing the plate on a shaker set to low speeds until a visible aggregate is formed in sample wells. The supernatant should be clear, olive green color. Note: Absence of aggregate after 2-3 minutes indicates very low DNA levels in the sample.

1. Place the sample on the Magnetic Separator for 30 seconds to 1 minute.
2. Without removing the plate from the Magnetic Separator, carefully remove and discard the supernatant using a 1 ml pipette without disturbing the pellet of beads by angling the pipette such that the tip is pointed away from the pellet.
3. Remove the plate from the Magnetic Separator.
4. Add 1 ml 50% (v/v) isopropanol to the samples and gently agitate the plate by swirling or placing the plate on a shaker set to low speeds for 15 seconds.
5. Place the sample on the Magnetic Separator for 30 seconds.
6. Without removing the plate from the Magnetic Separator, carefully remove and discard the supernatant using a 1 ml pipette without disturbing the pellet of beads.
7. Without removing the plate from the Magnetic Separator, add 150 µl Wash Buffer (W12) to the sides of the sample wells opposite to the bead pellet to ensure the bead pellet is not dislodged.
8. Incubate for 30 seconds at room temperature.
9. Without removing the plate from the Magnetic Separator, carefully remove and discard the supernatant using a 1 ml pipette without disturbing the pellet of beads.
10. Repeat Steps 8-10.
11. Proceed immediately to Eluting DNA.

 
Eluting DNA

1. Set a water bath to 65°C.
2. Remove the plate containing the pelleted magnetic beads (Step 11, above) from the Magnetic Separator.
3. Add 250 µl Elution Buffer (E5; 10 mM Tris-HCl, pH 8.5) to the samples and gently agitate the plate by swirling or placing the plate on a shaker set to low speeds to dislodge the pellet.
4. Incubate at 65°C for 30 minutes. For high DNA content, incubate the samples for an additional 30 minutes. 
5. Remove the plate from the water bath and gently agitate the plate by swirling or placing the plate on a shaker set to low speeds until the bead pellet is completely dispersed. Note: If the pellet is not completely dispersed tip-mix without forming bubbles. For samples with high DNA content, incubate the tube over night and tip-mix gently before proceeding to the next step. Tip: If the Elution Buffer is too viscous, add additional 150-250 µl Elution Buffer (E5) and keep the samples overnight at room temperature at this stage to increase the final yield.
6. Place the sample on the Magnetic Separator until the supernatant is clear and colorless (usually 15 minutes to 1 hour, viscous samples require longer time).
7. Without removing the plate from the Magnetic Separator, carefully remove the supernatant containing the DNA using a 1 ml pipette without disturbing the pellet of beads and transfer the supernatant to a sterile tube. Note: If the supernatant is discolored, repeat Steps 6-7.
8. Discard the used magnetic beads. Do not re-use the magnetic beads.

 
Storing DNA

• Store the purified DNA at -20°C or use DNA for the desired downstream application.
• To avoid repeated freezing and thawing of DNA, store the purified DNA at 4°C for immediate use or aliquot the DNA and store at -20°C for long-term storage

Introduction

Instructions for isolating gDNA from 3-10 ml human blood are described below. To isolate gDNA from 0.3-1 ml human blood.
 
The procedure is designed for isolating gDNA using the GeneCatcher™ Magnetic Beads procedure.
 
50 ml Tube Magnetic Separator

You will need a magnetic rack (separator) for use with the GeneCatcher™ gDNA 3-10 ml Blood Kit. We recommend using the 50 ml Tube Magnetic Separator (Catalog No. CS15050) for 50 ml tubes to obtain the best results. Other magnetic separators may not provide similar magnetic strength or may not be compatible with the volumes used in the protocol. The 50 ml Tube Magnetic Separator is a magnetic separation rack for use in protocols with magnetic beads. The 50 ml tube fits onto the magnetic rack associating the neodymium magnet with the tube for simple sample processing using magnetic beads (see figure below).



 
Materials Needed

• Blood sample (3-10 ml)
• 50 ml Tube Magnetic Separator
• 100% Isopropanol
• 50% (v/v) Isopropanol
• Sterile 1 x 50 ml tube per sample
• Sterile 1-, 5-, 10-ml pipettes
• Water bath or heat block set at 65°C
• Vortex mixer

 
Components supplied with the kit

• GeneCatcher™ Lysis Buffer (L13)
• Protease
• Protease Buffer
• GeneCatcher™ Magnetic Beads
• GeneCatcher™ Wash Buffer (W12)
• GeneCatcher™ Elution Buffer (E5)

 

Specific Reagent Volumes

Use the specified reagent volumes as indicated in the protocol based on the starting volume of the blood sample to obtain the best results. For some reagents, the same volume of reagent is used irrespective of the blood volume as indicated in the protocol.
 
To purify genomic DNA from 2 ml blood sample, use the specified reagent volumes for 3 ml blood samples as described in the protocol. To obtain the best results we recommend to use a starting blood volume of 3 ml.
 
Binding DNA

Follow the procedure below to bind DNA to the GeneCatcher™ Magnetic Beads.

Note:  For 2 ml blood samples, use the specified reagent volumes for 3 ml blood samples.

1. Vortex the tube containing the GeneCatcher™ Magnetic Beads to fully resuspend and evenly distribute the beads in the storage buffer.
2. Add resuspended GeneCatcher™ Magnetic Beads and Lysis Buffer (L13) to a sterile 50-ml tube. Mix the beads by gently swirling the tube until the beads are evenly distributed
   
   

   Blood (ml)	3	4	5	6	7	8	9	10
   Beads (µl)	45	60	75	90	105	120	135	150
   L13 (ml)	9	12	15	18	21	24	27	30

   
   
    
3. Add 2-10 ml well mixed blood samples to the tube containing the beads and gently invert the capped tube 3 times to mix the beads.
4. Incubate at room temperature for 5 minutes to allow the DNA to bind to the beads. During incubation, gently invert the tube occasionally for mixing.
5. Place the tube on a 50 ml Tube Magnetic Separator Rack (see above for a figure) for 3 minutes.
6. Without removing the tube from the Magnetic Separator, carefully remove and discard the supernatant using a 5 ml pipette without disturbing the pellet of beads by angling the pipette tip away from the pellet.
   
   TOP
7. Remove the tube from the Magnetic Separator.
8. Add 5 ml Lysis Buffer (L13) irrespective of the blood volume to the tube and gently invert the capped tube 3 times to mix the beads and wash away residual contaminants.
9. Incubate at room temperature for 30 seconds.
10. Place the tube on the Magnetic Separator for 20 seconds.
11. Without removing the tube from the Magnetic Separator, carefully remove and discard the supernatant using a 5 ml pipette without disturbing the bead pellet.
12. Proceed immediately to Protease Digestion.

 
Protease Digestion

1. Set a water bath to 65°C.
2. Remove the tube containing the pelleted magnetic beads from the Magnetic Separator (Step 11, above).
3. Add Protease Buffer and Protease to the tube and vortex the capped tube to thoroughly disperse the bead pellet (~30 seconds). Note: For 2 ml blood samples, use the specified reagent volumes for 3 ml blood samples.
   
   

   Blood (ml)	3	4	5	6	7	8	9	10
   Protease Buffer (ml)	3	3	3	3	3.5	4	4.5	5
   Protease (µl)	40	40	40	40	40	40	40	40

   
   
    
4. Incubate the tube at 65°C for 10 minutes and then cool to room temperature (~10-20 minutes).
5. Gently invert the tube twice to resuspend any settled beads.
6. Proceed immediately to Washing DNA.

 
Washing DNA

1. Add 100% isopropanol (IPA) to the tube and mix by gently inverting the tube until a visible aggregate forms. Note:  For 2 ml blood samples, use the specified reagent volumes for 3 ml blood samples.
   
   

   Blood (ml)	3	4	5	6	7	8	9	10
   IPA (ml)	3	3	3	3	3.5	4	4.5	5

   
   Absence of aggregate after 2-3 minutes indicates very low DNA levels in the sample.
2. Place the sample on the Magnetic Separator for 30 seconds to 1 minute.
3. Without removing the tube from the Magnetic Separator, carefully remove and discard the supernatant using a 5 ml pipette without disturbing the pellet of beads.
4. Remove the tube from the Magnetic Separator.
5. Add 3 ml 50% (v/v) isopropanol irrespective of the blood volume to the tube and mix by gently inverting the capped tube 5 times.
6. Place the sample on the Magnetic Separator for 30 seconds.
7. Without removing the tube from the Magnetic Separator, carefully remove and discard the supernatant using a 5 ml pipette without disturbing the pellet of beads.
8. Keep the tube on the Magnetic Separator for an additional 1 minute to allow any remaining liquid to settle to the bottom of the tube. Remove and discard the liquid using a 1-ml pipette.
9. Without removing the tube from the Magnetic Separator, add 250 µl Wash Buffer (W12) irrespective of blood volume to the sides of the tube wells opposite to the bead pellet to ensure the bead pellet is not dislodged.
10. Incubate for 1 minute at room temperature.
11. Without removing the tube from the Magnetic Separator, carefully remove and discard the supernatant using a 1 ml pipette without disturbing the bead pellet.
12. Repeat Steps 9-11.
13. Proceed immediately to Eluting DNA.

 

Eluting DNA

1. Set a water bath to 65°C.
2. Remove the tube containing the pelleted magnetic beads (Step 12, above) from the Magnetic Separator.
3. Add 1 ml Elution Buffer (E5; 10 mM Tris-HCl, pH 8.5) irrespective of the blood volume to the tube and gently agitate the sample by swirling the tube to release the bead pellet from the tube wall. For samples with high DNA content.
4. Incubate at 65°C for 1 hour. For high DNA content, incubate the samples for an additional 30 minutes.
5. Remove the tube from the water bath and gently swirl the tube until the bead pellet is completely dispersed.
   
   Note:  If the pellet is not completely dispersed tip-mix without forming bubbles. For samples with high DNA content, incubate the tube over night and tip-mix gently before proceeding to the next step. Tip: If the Elution Buffer is too viscous, add additional 0.5-1 ml Elution Buffer (E5) and keep the samples overnight at room temperature at this stage to increase the final yield.
6. Place the tube on the Magnetic Separator until the supernatant is clear and colorless (usually 15 minutes to 1 hour, viscous samples require longer time).
7. Without removing the tube from the Magnetic Separator, carefully remove the supernatant containing the DNA using a 1 ml pipette without disturbing the pellet of beads and transfer the supernatant to a sterile tube. Note: If the supernatant is discolored, repeat Steps 6-7.
8. Discard the used magnetic beads. Do not re-use the magnetic beads.

Storing DNA

• Store the purified DNA at -20°C or use DNA for the desired downstream application.
• To avoid repeated freezing and thawing of DNA, store the purified DNA at 4°C for immediate use or aliquot the DNA and store at -20°C for long-term storage.

1. Prepare a dilution of the DNA solution in 10 mM Tris-HCl, pH 7.5. Mix well. Measure the absorbance at 260 nm (A₂₆₀) of the dilution in a spectrophotometer (using a cuvette with an optical path length of 1 cm) blanked against 10 mM Tris-HCl pH 7.5


2. Calculate the concentration of DNA using the formula:
   DNA (µg/ml) = A₂₆₀ x 50 x dilution factor
   For DNA, A₂₆₀ = 1 for a 50 µg/ml solution measured in a cuvette with an optical path length of 1 cm.

Introduction

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