Blood DNA
 
 DNA/RNA Extraction Kits
 Orochem offers a complete line of kits in individual spin columns and 96-well formats for DNA/RNA extraction from a variety of sample sources such as Bacteria, plant, blood, serum, tissue and forensic samples.
 From Bacteria (Plasmid, Genomic): High yield of Plasmid DNA in less than 20 minutes
 From Blood: Available in a spin column or 96 well formats for blood or serum.
 From Tissue: For all kinds of tissues including mouse tails. Fast protocol and methodology eliminates hands-on homogenization plus no hazardous organic solvents required.
 From Plant: Completely removes all polysaccharide and phenolic inhibitors of PCR from plant material. Can be used with seeds, leaves, bark and roots.
 Forensic Applications:
  Tel.: 630‐916‐0225 Fax: 630‐916‐0250  www.orochem.com  Orochem Technologies Inc 331 Eisenhower Ln South,  Lombard, IL 60148
DNA for Forensic Applications!
GenQuikTM Blood DNA Extraction Kit (Whole blood, Buffy coat, lymphocytes, plasma. Serum and Body fluids) Catalog # OGQDNAB-50
 Introduction GenQuikTM Blood DNA Extraction Kit is ideal for rapid isolation of an average of 6 μg of total DNA (e.g., genomic, viral, mitochondrial) from 250 μl of whole human blood, and up to 50 μg of DNA from 250 μl of buffy coat, 5 x 106 lymphocytes, or cultured cells that have a normal set of chromosomes. The method is suitable for use with whole blood treated with citrate, heparin, or EDTA. Samples may be either fresh or frozen. This method is based on the efficient release of genomic DNA from anti-coagulated whole blood by a special cell lysis and heme/protein precipitation buffer (Buffer DBL2) coupled with the selective adsorption of the genomic DNA to a special miniPrep column due to the high salt nature of the buffer. The filter is washed. As a result, the DNA is recovered in certified DNAase-free Tris buffer.
 
Features and Benefits For DNA purification from     • Whole blood    • Plasma    • Serum    • Buffy coat    • Body fluids • Lymphocytes   • Cultured cells    • Swabs    • Dried blood spots
 Rapid Protocol – Rapid DNA isolation from various sources. High Quality - Yields DNA greater than 50 kb in length. DNA is ready for PCR, restriction digests, and all downstream applications. Flexible Sizes - Use GenQuikTM Blood Spin Kit for 200-300 μl whole blood and GenQuikTM Mega Blood Spin Kit for  10 ml of whole blood.
 Amounts of starting material for GenQuikTM Mini procedures  Sample Amount Blood, plasma, serum –250 μl Buffy coat- 250 μl Cells (diploid) 5 x 106 cells Small samples should be adjusted to 250 μl with PBS before loading, while for samples larger than 300 μl, the amount of lysis buffer and other reagents added to the sample before loading must be increased proportionally (see note below). Application of the lysed sample to the GenQuikTM Spin Column will require more than one loading step if the initial sample volume is
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  Tel.: 630‐916‐0225 Fax: 630‐916‐0250  www.orochem.com  Orochem Technologies Inc 331 Eisenhower Ln South,  Lombard, IL 60148
increased. The amounts of Buffer DBL3 and Buffer DBLW used in the wash steps need not be increased. It is important not to overload the column, as this can lead to significantly lower yields than expected.
 
Determination of yield and purity DNA yield is determined by measuring the concentration of DNA in the elute by its absorbance at 260 nm. Absorbance readings at 260 nm should fall between 0.1 and 1.0 to be accurate. An A 260 of 1 (with a 1 cm detection path) corresponds to 50 μg DNA per milliliter water. Water should be used as diluents when measuring DNA concentration since the relationship between absorbance and concentration is based on extinction coefficients calculated for nucleic acids in water. Volume of DNA sample = 100 μl Dilution = 20 μl of DNA sample + 180 μl distilled water (1/10 dilution)
 Complete - All tubes and reagents required are included. Low to High Throughput - GenQuikTM Blood Spin Kit is also available in a 96 well plate format.
 
PRECAUTION Please wear gloves as working with this product. Avoid all skin contact with reagents in this kit. In case of contact, wash thoroughly with water. Reagents labeled flammable should be kept away from open flames and fire. Avoid contact with bleach or other oxidizers. This kit is for research purposes only. Not for diagnostic use.
 Equipment required; but not included 1. Micro centrifuge (13,000 x g) 2. 65°C water bath 3. Pipettes (volumes required 20 μl - 500 μl), vortex
 
Kit Contents
 Component Description Suspension Buffer (DBL1) Cell lysis buffer Lysis Buffer (DBL2) Protein-depleting buffer Binding Buffer (DBL3) Binding Buffer Wash Buffer (DBLW) Add amount of ethanol specified and store at room temperature. Either 100% or 95% denatured ethanol can be used
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  Tel.: 630‐916‐0225 Fax: 630‐916‐0250  www.orochem.com  Orochem Technologies Inc 331 Eisenhower Ln South,  Lombard, IL 60148
Elution Buffer (DBLE) 5 mM Tris-HCl, 0.1 mM EDTA, pH 8.5. Store at room temperature Miniprep column 
 
Protocol manual The buffers supplied in this kit are stable for a period of at least 12 months from the date of receipt when stored under ambient conditions. Please avoid exposure to extremes in temperature or direct sunlight.
 
CAUTION Buffer DBL1 and Buffer DBL2 contain chemical irritants. When working with these buffers, always wear suitable protective clothing such as safety glasses, laboratory coat and gloves. Be careful to avoid contact with eyes and skin. In the case of such contact, wash immediately with water. If necessary, seek medical assistance.
 
Preparation before experiment Before using the kit, add the amount of ethanol specified on the bottle label to the Buffer DBLW concentrate and mix well. Either 100% or 95% (denatured) ethanol can be used.
 
Detailed Protocols The Mini-prep column has a maximum binding capacity of approximately 25μg. If more genomic DNA is required, process 0.5 ml of whole blood in the following manner. To extract DNA from 0.5 ml of blood, divide the sample into 2×250 μl aliquots and prepare extracts by following Steps 1-4 in two separate Microfuge tubes. Combine the supernatant obtained in step 4 into one Miniprep column to consolidate the genomic DNA and increase the yield. Elute the purified genomic DNA in 100-200μl of Buffer TE.  For maintaining the integrity and reactivity of the genomic DNA, particularly in PCR, the purified genomic DNA should be eluted and stored in low-salt Tris buffer containing 0.5-1 mM EDTA (provided). We recommend the use of nucleic acid-free and nuclease-free plasticware. 1. Add 500 μl of Buffer DBL1 to a 1.5 ml microfuge tube. 2. Add 200-250 μl of anti-coagulated whole blood. Close the cap of the Microfuge tube and mix by vortexing at top speed for 10 seconds. Note: Vortexing is required for complete release of the genomic DNA. Although vortexing will result in limited shearing of the genomic DNA, it will have no effect upon the performance of the genomic DNA in applications which require high molecular DNA.
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  Tel.: 630‐916‐0225 Fax: 630‐916‐0250  www.orochem.com  Orochem Technologies Inc 331 Eisenhower Ln South,  Lombard, IL 60148
Note: To extract genomic DNA from clotted or dried blood, place the sample in a mortar (ambient temperature) and add 200 μl of 20 mM Tris, 10 mM EDTA, pH 8.5. Grind rapidly for 30 seconds to disperse the sample. Add 500 μl of Buffer DBL1 pre-heated to 50°C and grind briefly or pipette to dissolve the sample. Transfer the sample to a 1.5 ml Microfuge tube with a transfer pipette or other device. Vortex for 10 seconds to further dissolve the dried or clotted blood. Note: If blood volume is <200 μl, add deionized water or PBS to 200 μl. 3. Add 400 μl of Buffer DBL2 and mix by vortexing at top speed for 10 seconds. 4. Centrifuge at 12,000×g for 10 minutes at ambient temperature to pellet cellular debris. 5. Place a Miniprep column into a 2 ml Microfuge tube. Pipette the clarified supernatant obtained from step 4 into the Miniprep column. Centrifuge at 6,000×g for 1 minute. 6. Discard the filtrate from the 2 ml Microfuge tube. Place the Miniprep column back into the 2 ml Microfuge tube. Pipette 500 μl of Buffer DBL3 into the Mini-prep column and allow standing at room temperature for 2 minutes. Centrifuge at 6,000×g for 1 minute. Note: If any liquid remains in the Miniprep column after centrifugation, extend the centrifuge time or increase the g-force. 7. Discard the filtrate from the 2 ml Microfuge tube. Place the Miniprep column back into the 2 ml Microfuge tube. Add 600 μl of Buffer DBLW to the Miniprep column and centrifuge at 12,000×g for 1 minute. Note: Make sure that ethanol has been added into Buffer DBLW concentrate. 8. Optional Step: Discard the filtrate from the 2 ml Microfuge tube. Place the Miniprep column back into the 2 ml Microfuge tube. Add 500 μl of Buffer DBLW to the Miniprep column and centrifuge at 12,000×g for 1 minute. Note: Two washes with Buffer DBLW are used to ensure the complete removal of salt, eliminating potential problems in subsequent enzymatic reactions. 9. Discard the filtrate from the 2 ml Microfuge tube. Place the Miniprep column back into the 2 ml Microfuge tube. Centrifuge at 12,000×g for 1 minute. 10. Place the Miniprep column into a 1.5 ml Microfuge tube. Add 80-200 μl of Buffer DBLE. Allow to stand at room temperature for 1 minute. Centrifuge at 12,000×g for 1 minute to elute the genomic DNA.
 
 
 
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  Tel.: 630‐916‐0225 Fax: 630‐916‐0250  www.orochem.com  Orochem Technologies Inc 331 Eisenhower Ln South,  Lombard, IL 60148
 
Troubleshooting 1. Low or no yield ƒ Low WBC content in blood sample ƒ Inefficient lysis with Buffer DBL1 ƒ Inefficient mixing with Buffer DBL2 ƒ DNA not efficiently eluted ƒ Blood coagulation 2. Low A260/280 ƒ Inefficient lysis with Buffer DBL1 ƒ Inefficient mixing with Buffer DBL2 ƒ Buffers DBL1 and DBL2 used in the wrong order ƒ Blood has been stored at room temperature for extended periods 3. Genomic DNA appears to be degraded Depending upon the completeness of degradation, the genomic DNA will either appear as a diffuse smear or as a smear trailing in front of a high molecular weight band on an agarose gel. Since no physical measure used during the purification process is sufficient to cause any visually discernable degradation, the most likely source is enzymatic. Enzymatic degradation may result from prolonged or improper storage of the blood sample. 4. Genomic DNA performs poorly in enzymatic reactions ƒ Low DNA concentration ƒ Salt contamination ƒ Ethanol contamination 5. Clogged spin-filter ƒ Elevated WBC content in blood sample ƒ Inefficient lysis with Buffer DBL1 ƒ Inefficient mixing with Buffer DBL2 ƒ Blood was insufficiently mixed after phlebotomy, resulting in coagulation ƒ Precipitates have formed in blood that has been stored either froz