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Blood genomic DNA kit
The blood gDNA kit provides an easy method for isolating genomic DNA from fresh, frozen or anticoagulated whole blood. This method is also suitable for isolating genomic DNA from buffy coat, plasma serum and Buccal Swab. The purified DNA is ready for downstream applications such as PCR, Southern blotting and RFLP.
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Blood gDNA mini kit
Cat#: GD2311-01 Size: 50
Isolation form?<1mL blood samples up to 2~30 microgram DNA, including viral genomic DNA Learn More
$ 90.00
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Blood gDNA mini kit
Cat#: GD2311-02 Size: 250
Isolation form?<1mL blood samples up to 2~30 microgram DNA, including viral genomic DNA Learn More
$ 420.00
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96-well blood DNA kit
Cat#: GD2815-01 Size: 4x96
96 well blood genomic DNA purification kit Learn More
$ 680.00
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96-well blood DNA kit
Cat#: GD2815-02 Size: 20x96
96 well blood genomic DNA purification kit Learn More
$ 3,000.00
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Introduction The EZgeneTM family of products is an innovative system that simplifies the extraction and purification of nucleic acids from a variety of sources. The key to this system is the new ezBind matrix that specifically, but reversibly, binds DNA or RNA under certain optimal conditions allowing proteins and other contaminants to be removed. Nucleic acids are easily eluted with deionized water or a low salt buffer. The EZgeneTM Blood gDNA Kit provides an easy and rapid method for the isolation of genomic DNA for consistent PCR and Southern analysis. Up to 250 μL of fresh, frozen or anticoagulated whole blood can be readily processed at one time. This gDNA Kit can also be used for the preparation of genomic DNA from buffy coat, serum, plasma, saliva, buccal swab and other body fluids. The EZgeneTM Blood gDNA Kit allows for the single or multiple simultaneous processing of samples. There is no need for phenol/chloroform extractions, and time consuming steps are eliminated (e.g. precipitation using isopropanol or ethanol). Purified DNA obtained with the EZgeneTM Blood gDNA Kit will be ready for applications such as PCR, Southern Blotting, and Restriction Digestion.
Storage and Stability
All EZgeneTM Blood gDNA Kit components are guaranteed for at least 12 months from the date of purchase when stored as follows: Reconstituted Protease K at - 20 ℃, and all other materials at RT (22-25 ℃). Under cool ambient conditions, a precipitate may form in Buffer BL. In case of such an event, heat the bottle at 37 ℃ to dissolve.
Binding Capacity
Each ezBind column can bind approximately 100 μg DNA. Using greater than 250 μL of whole Blood or of buffy coat is not recommended.
Kit content
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Product |
GD2311-00 |
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Purification Times |
4 preps |
50 preps |
250 preps |
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ezBind DNA Mini Columns |
4 |
50 |
250 |
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2 mL Collection Tubes |
12 |
150 |
750 |
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Buffer BL |
2.5 mL |
28 mL |
140 mL |
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Buffer KB |
2.5 mL |
30 mL |
140 mL |
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DNA Wash Buffer |
3 mL |
30 mL |
3 x 36 mL |
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Elution Buffer* |
2 mL |
30 mL |
140 mL |
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Protease K |
2 mg |
30 mg |
5 x 30mg |
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RNase A |
45 μL |
510 μL |
2 x 1.4 mL |
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Instruction Booklet |
1 |
1 |
1 |
Elution Buffer = 10 mM Tris-HCl, pH 8.5 * Buffer BL contains Chaotropic salts. Equilibration Buffer contains Sodium Hydroxide. Use gloves and protective eyeware when handling this solution.
Before Starting
It is strongly advised that you familiarize yourself with the entire booklet before starting. EZgeneTM Kits are designed to be simple, fast, and reliable provided that all steps are followed diligently.
Important Reconstitute
Each vial of Protease K 110 μL (4 preps), 1.3 mL (50 preps), or 6.5 mL (250 preps) of Elution Buffer. Vortex the vial briefly before use. We recommend that you aliquot, and store vials of reconstituted Protease K at -20 ℃. Dilute DNA Wash Buffer with absolute ethanol as follows: GD2311-00: Add 7 mL of absolute ethanol to each bottle GD2311-01: Add 70 mL of absolute ethanol to each bottle GD2311-02: Add 84 mL of absolute ethanol to each bottle
EZgeneTM Blood DNA Isolation Protocols IA.
Blood & Body Fluid DNA Protocol Materials provided by user
Tabletop microcentrifuge
Sterile 1.5mL centrifuge tubes
Water bath
RNase stock solution
Absolute ethanol
PBS Buffer
All centrifugation steps must be carried out at room temperature
NOTE: The procedure below has been optimized for the use with FRESH or FROZEN blood samples of 1~250 μL in volume. Anticoagulated Blood, Saliva, Serum, Buffy Coat or other Body Fluids can also be used. In addition, # 107 of leukocytes or cultured cells may be used with this procedure. For DNA extraction from tissue and mouse tail we suggest that you use the EZgeneTM Tissue gDNA Kit. To isolate Viral RNA from serum or other non-cellular body fluids we recommend using the EZgeneTM Viral RNA Kit.
- 1. Transfer the sample into a sterile microcentrifuge tube and bring the volume up to 250 μL with 10 mM Tris-HCl, PBS, or Elution Buffer (provided).
- 2. Add 25 μL of Reconstituted Protease K and 250 μL of Buffer BL. Vortex at maximum speed for 15 s to mix thoroughly. If RNA-Free Genomic DNA is required, add 10 μL of RNase A (20mg/mL) to each sample.
- 3. Incubate the sample at 65 ℃ for 10 min. BRIEFLY vortex the tube once while incubation.
Note: During incubation step 5 can be processed.
- 4. Add 260 μL of absolute ethanol (RT, 96-100%) to lysate. Vortex at maximum speed for 20 s. Briefly centrifuge the tube to collect any drops from the inside of the lid.
- 5. Transfer the lysate from step 4 into the column, and centrifuge at 10,000 x g for 1 min to bind DNA. Discard the collection tube and flow-through liquid.
- 6. Place the column into a new provided 2 mL collection tube. Add 500 μL of Buffer KB, and centrifuge at maximum speed for 30 s. Discard collection tube, and flow through liquid.
- 7. Place the column into the same 2 mL collection tube from step 7, and add 700 μL of DNA Wash Buffer. Centrifuge at 10,000 x g for 1 min. Discard collection tube, and flow-through liquid.
NOTE: DNA Wash Buffer is provided as a concentrate and must be diluted with absolute ethanol as indicated on the bottle, and on page 3. If refrigerated, the diluted DNA Wash Buffer must be brought to room temperature before use.
- 8. Using a new 2 mL collection tube, repeat step 7 with an additional 600 μL of DNA Wash Buffer diluted with absolute ethanol, and centrifuge as above. Discard flow through. (Collection tube will be re-used in the following step)
- 9. Place the empty column into the same 2 mL collection tube, with the lid open, from step 8, centrifuge at a maximum speed of (13,000 x g) for 2 min to dry the column.
This step is crucial for ensuring optimal elution in the following step.
- 10. Place the column into a sterile 1.5 mL microfuge tube, add 100-200 μL of preheated (65 ℃) Elution Buffer (10 mM Tris-HCl, pH 8.5). Allow sit at room temperature for 5 min.
- 11. To elute DNA from the column, centrifuge at (13,000 x g) for 1 min. Retain flow-through containing the DNA. Place the column into a new 1.5 mL tube. Elute DNA again as indicated in previous step. Discard column, and store the Eluted DNA at -20 ℃
IB: Blood & Body Fluid DNA Vacuum/Spin Protocol
- 1. Prepare the lysate and column by following steps 1-4 of the Spin Protocol.
- 2. Insert an ezBind DNA Mini Column into the vacuum manifold. CAREFULLY apply the lysate to the column. Turn on the vacuum source to draw all of the liquid through the column. Turn off the vacuum.
NOTE: If at this stage the lysate has difficulty passing through the column, place the column into a collection tube (supplied). Close the lid and centrifuge at 13,000 x g for 2 min or until all of the liquid has passed through. Place the column into another collection tube (supplied), and proceed to step 6 of the spin protocol on page 5.
- 3. Add 500 μL of Buffer HB into the column. Turn on the vacuum source to draw all of the liquid through the column. Turn off the vacuum.
- 4. Wash the column by pipetting 700 μL of DNA Wash Buffer diluted with ethanol into the column. Turn on the vacuum source to draw all of the liquid through the column. Turn off the vacuum.
- 5. Repeat Step 4 with another 600 μL of DNA Wash Buffer.
- 6. Remove the column from the vacuum manifold. Insert the column into a collection tube, with the lid open, and centrifuge at maximum speed (13,000 x g) for 2 min to completely dry the column.
- 7. Place the Column into a sterile 1.5 mL microfuge tube and add 100-200 μL of preheated (65 ℃) Elution Buffer (10 mM Tris-HCl, pH 8.5). Allow tubes to sit for 5 min at room temperature.
- 8. To elute DNA from the column, centrifuge at 13,000 x g for 1 min. Retain flow through containing the DNA. Place the column into a second 1.5 mL tube. Elute DNA again as indicated in the previous step. Discard column, and store the Eluted DNA at -20 ℃
NOTES ON ELUTION
The first elution will typically produce yields of 60-70% of the DNA bound to the column. Thus two elutions will generally give > 90% yields. However, increasing the elution volume will also reduce the concentration of the final product. To obtain DNA at higher concentrations, the elution can be carried out using 50 μL - 100 μL of Elution Buffer (which slightly reduces overall DNA yield). Volumes lower than 50 μL greatly reduce yields. In some instances yields may be increased by incubating the column at 70 ℃ (rather than at room temperature) upon the addition of Elution Buffer. If necessary the DNA can be concentrated. Add sodium chloride to reach a final concentration of 0.1 M followed by 2x volume of absolute (~96-100%) ethanol. Mix well and incubate at –20℃ for 10 min. Centrifuge at 10,000 x g for 15 min and discard supernatant. Add 700 μL of 70% ethanol and centrifuge at 10,000 x g for 2 min. Discard supernatant, air dry the pellet for 2 min, and resuspend the DNA in 20 μL of sterile deionized water or 10 mM Tris-HCl, pH 8.5. The expected yield from 250 μL of blood is approximately 4-12 μg of DNA.
IIA. Buccal Swab DNA Spin Protocol
IMPORTANT NOTE: This protocol requires an increased volume of Buffer BL; fewer preparations can be performed. Additional Buffer BL can be purchased separately. All centrifugation steps must be carried out at room temperature
- 1. Place the Buccal Swab in a 2 mL centrifuge tube and add 500 μL of PBS Buffer to the tube. If RNA-free DNA is required for downstream applications, add 8 μL of RNase A Stock Solution (20mg/mL) into the sample.
- 2. Add 25 μL of Reconstituted Protease K and 500 μL of Buffer BL. Vortex at maximum speed for 30 s to mix thoroughly. Incubate the sample at 65 ℃ for 10 min.
Note: step 5 can be processed while incubating sample.
- 3. Remove the Buccal Swab.
- 4. Add 500 μL of absolute ethanol (room temperature, 96-100%) to the sample, and mix by vortexing. Collect any liquid drops remaining on the lid by briefly centrifugation.
- 5. CAREFULLY transfer 750 μL of the sample from step 4 into the column, and centrifuge at 10,000 x g for 1 min. Discard the collection tube and flow-through liquid.
- 6. Place the Column into a new provided 2 mL collection tube. CAREFULLY apply the remainder of the sample from step 4 to the column. Centrifuge as above. Discard collection tube, and flow-through liquid.
- 7. Place the Column into a new 2 mL collection tube. Add 500 μL of KB Buffer to the column. Centrifuge as above. Discard the flow-through and reuse the collection tube in the following step.
- 8. Place the ezBind DNA Mini Columns into the same 2 mL collection tube from step 7, and add 700 μL of DNA Wash Buffer diluted with ethanol. Centrifuge as above. Discard flow-through liquid, and reuse the collection tube in the following step.
NOTE: DNA Wash Buffer is provided as a concentrate and must be diluted with absolute ethanol as indicated on the bottle, and on page 3. If refrigerated, the diluted DNA Wash Buffer must be brought to room temperature before use.
- 9. REPEAT wash step 8 with another 600 μL of DNA Wash Buffer diluted with ethanol.
- 10. Place the column into the same 2 mL collection tube from step 9. Centrifuge at 13,000 x g for 2 min to completely dry the ezBind DNA Mini Columns Discard the flow-through and the collection tube.
- 11. Place the column into a sterile 1.5 mL microcentrifuge tube, and add 100-200 μL of preheated Elution Buffer (10 mM Tris-HCl, pH 8.5, 65 ℃). Allow tubes to sit for 5 min at room temperature.
- 12. To elute DNA from the column, centrifuge at maximum speed for 1 min. Retain flow through containing the DNA. Place the column into a second 1.5 mL tube. Elute DNA again as described above. Discard column, and store the Eluted DNA at -20 ℃.
IIB. Buccal Swab DNA Vacuum/Spin Protocol
- 1. Prepare Buccal Swab Lysate by following steps 1-4 of the Spin Protocol on page 8.
- 2. Insert the column into the vacuum manifold. Carefully, apply the lysate to the column. Turn on the vacuum source to draw the lysate from step 4 of the Spin Protocol on page 6 through the column. Turn off the vacuum. REPEAT this step until all of the lysate has passed through the column.
- 3. Pipet 500 μL of Buffer KB into the column. Turn on the vacuum source to draw all of the liquid through the column. Turn off the vacuum.
- 4. Wash the column by pipetting 700 μL of DNA Wash Buffer diluted with ethanol into the column. Turn on the vacuum source to draw all of the liquid through the column. Turn off the vacuum.
- 5. REPEAT wash step 4 with another 600 μL of DNA Wash Buffer.
- 6. Remove it from the vacuum manifold. Insert the column, with the lid open, into a collection tube (supplied), and centrifuge at 13,000 x g for 2 min to completely dry the column.
- 7. Proceed with steps 9 and 11 of the Spin Protocol on page 8
III. Dried Blood DNA Spin Protocol
- 1. Cut or punch out the blood spot from the filter paper (up to 200 μL of blood can be used per spot). Tear or cut the filter paper into small pieces and place them into a microfuge tube.
- 2. Add 250 μL of PBS Buffer, and incubate at 65 ℃ for 1 hr, while vortexing every 20 min for proper mixing.
- 3. Add 25 μL of Protease K stock solution and mix well. Incubate for 30 min at 65 ℃ with occasional mixing.
- 4. Centrifuge at 13,000 x g for 5 min at room temperature.
- 5. Transfer the supernatant to a clean microfuge tube and add 1 volume of Buffer BL followed by 1 volume of absolute ethanol. Vortex thoroughly .Collect any drops remaining on the lid by briefly centrifuging.
- 6. Transfer the lysate from step 5 into the column, and centrifuge at 10,000 x g for 1 min to bind the DNA. Discard the flow-through liquid and the collection tube.
- 7. Place the Column into a new 2 mL collection tube. Add 500 μL of Buffer KB to the column, and centrifuge as above. Discard the flow-through and reuse the collection tube in the following step.
- 8. Place the column into the same 2 mL collection tube from step 7, and wash by pipetting in 700 μL of DNA Wash Buffer diluted with ethanol. Centrifuge as above. Dispose of the collection tube and flow-through liquid.
NOTE: DNA Wash Buffer is provided as a concentrate and must be diluted with absolute ethanol as indicated on the bottle, and on page 4. If refrigerated, the diluted DNA Wash Buffer must be brought to room temperature before use.
- 9. Using a NEW collection tube (provided), wash the column with a second 600 μL of DNA Wash Buffer diluted with ethanol. Centrifuge as above. Discard flow-through liquid, and reuse the collection tube in the following step.
- 10. Place the empty column, with the lid open, into the same 2 mL collection tube from step 9. Centrifuge at maximum speed (13,000 x g) for 2 min to
- completely dry the column. Discardf the flow-through and the collection tube.
NOTE: This step is crucial for ensuring optimal elution in the following steps.
- 11. Place the column into a sterile 1.5 mL microcentrifuge tube, and add 100-200 μL of preheated Elution Buffer (10 mM Tris-HCl, pH 8.5, 65 ℃). Allow tubes to sit for 5 min at room temperature.
- 12. To elute DNA from the column, centrifuge at maximum speed (15,000 x g) for 1 min. Retain flow through containing the DNA. Discard column, and store the Eluted DNA at -20℃.
NOTE: Blood Spots from finger pricks usually contain no more than 50 μL of blood, and yield approximately 500 ng to 1 μg of DNA. This is usually sufficient for PCR analysis. To obtain higher DNA concentrations, elute with 50 μL of preheated elution buffer (volumes lower than 50 μL greatly reduce yields). Alternatively, the first eluate can be used to perform second elution.
IV. Buffy Coat DNA Spin Protocol The buffy coat fraction of whole blood is enriched with WBC’s, and usually gives at least 5-folds more of DNA than the same volume of blood. To prepare buffy coat from fresh whole blood, simply centrifuge the sample at 3,000 - 4,000 x g for 10 min at room temperature. Three layers should be obtained with plasma in the upper layer, leukocytes in the middle layer (buffy coat), and erythrocytes in the bottom layer. CAREFULLY aspirate the plasma while making sure not to disturb the layer of concentrated leukocytes. The buffy coat can be drawn off with a pipette, and be used directly in the EZgeneTM Blood gDNA Protocol, or frozen at -70℃ for storage.
Determination of Yield and Quality The total DNA yield can be determined by a spectrophotometer using deionized water, Tris-HCl Buffer, or Elution Buffer as a blank. Dilute the DNA in TE buffer and calculate the concentration using the following equation: [DNA] = (Absorbance 260) x (0.05 μg/μL) x (Dilution Factor) The quality of DNA can be assessed by measuring absorbance at both 260 nm and at 280 nm. A 260/ 280 (A /A ) ratio of 1.7-1.9 corresponds to 85%-95% purity. Expected yields will range from 4 μg - 12 μg of DNA per 250 μL of whole blood, depending on the source of the sample, its age, and the method of storage. Yields are generally 5-fold higher with buffy coat samples.
Trouble Shooting Guide
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Problem |
Cause |
Possible Solution |
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Clogged Column |
Incomplete Lysis |
Add the correct volume of Buffer BL and incubate for specified time at 65℃. It may be necessary to extend incubation time by 10 min. |
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Sample is too Large |
If using more than 250μL of Blood, increase volumes of Protease, Buffer BL, and Isopropanol. Pass aliquots of lysate through one column successively. |
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Sample is too viscous |
Divide sample into multiple tubes, and adjust the volume to 250 μL with 10 mM Tris-HCL. |
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Low DNA Yield |
Clogged Column |
See above |
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Poor elution |
Repeat elution or increase elution volume (see notes on elution on page 7) Incubation of column at 70℃ for 5 min with Elution Buffer may increase yields. |
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Improper washing |
Wash Buffer Concentrate must be diluted with absolute (100%) ethanol as specified on page 3 before use. |
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Buffy Coat Used |
With Buffy Coat samples, use absolute ethanol, rather than isopropanol. |
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260 /280Low A /A Ratio |
Extended centrifugation during elution. |
Resin from the column may be present in eluate. Avoid centrifugation at speeds higher than specified. The material can be removed from the eluate by centrifugation-it will not interfere with PCR or restriction digests. |
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Poor cell lysis due to incomplete mixing with Buffer BL. |
Repeat the procedure, this time making sure to vortex the sample with Buffer BL immediately and completely. |
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Hemoglobin Remains on column |
After application of sample to the column, wash once with 300 μL of Buffer BL. |
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No DNA Eluted |
Poor Cell Lysis due to improper mixing with Buffer BL |
Mix thoroughly with Buffer BL prior to loading the column. |
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Absolute Ethanol not added to Buffer BL. |
Before applying the sample to the column and aliquot of Buffer BL/ethanol solution must be added. |
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No Ethanol added to Wash Buffer Concentrate |
Dilute Wash Buffer with the indicated volume of absolute ethanol before use (page 3). |
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Washing Leaves Colored Residue in Column |
Incomplete Lysis due to improper mixing with Buffer BL. |
Buffer BL is viscous and the sample must be mixed thoroughly |
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No Ethanol added to Wash Buffer Concentrate |
Dilute Wash Buffer with the indicated volume of absolute ethanol before use. |
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Eluted Material has Red/Brown Color |
Sample Volume is too Large |
Reduce sample volume, and proceed with protocol. |
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Hemoglobin remains in column |
After applying sample, wash column once with 300μL of Buffer BL. |
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