GenElute™ Blood Genomic DNA Kit Protocol

Product Description

Our GenElute™ Blood Genomic DNA Kit provides a simple and convenient way to isolate pure genomic DNA from fresh or aged (older than 24 hours) whole blood. The kit combines the advantages of silica binding with a microspin format, and eliminates the need for expensive resins, alcohol precipitation, and hazardous organic compounds such as phenol and chloroform. The starting material is lysed in a chaotropic salt-containing solution to insure the thorough denaturation of macromolecules. The addition of ethanol causes the DNA to bind when the lysate is spun through a silica membrane in a microcentrifuge tube. A Prewash Solution is provided to help remove contaminants that are associated with aged (older than 24 hours) whole blood samples. After washing to remove contaminants, the DNA is eluted in 200 µL of a Tris-EDTA solution.

The expected yields of genomic DNA will vary depending on the amount and nature of the starting material used (for example, 4–10 µg of RNase A-treated DNA can be isolated from 200 µL of fresh whole blood in less than one hour). DNA purified with this kit has an A₂₆₀/A₂₈₀ ratio between 1.6 and 1.9 and can be up to 50 kb in length. This DNA is ready for downstream applications such as restriction endonuclease digestions, PCR, Southern blots, and sequencing reactions.

Reagents Provided	Catalog No.	NA2010 70 Preps	NA2020 350 Preps
Resuspension Solution	P3980	20 mL	100 mL
Lysis Solution C	B8803	20 mL	90 mL
Column Preparation Solution	C2112	60 mL	225 mL
Prewash Solution Concentrate	P6365	22.5 mL	90 mL
Wash Solution Concentrate	B6553	20 mL	90 mL
Elution Solution (10 mM Tris-HCl, 0.5 mM EDTA, pH 9.0)	B6803	35 mL	180 mL
Proteinase K	P2308	3 x 10 mg	2 x 100 mg
RNase A Solution	R6148	1.7 mL	8 mL
GenElute™ Miniprep Binding Columns in Tubes	C9471	70 each	5 x 70 each
Collection Tubes, 2.0 mL capacity	T5449 or T7813	3 x 70 each	15 x 70 each

Equipment and Reagents Required But Not Provided

• 55 °C water bath or shaking water bath
• Pipette tips with aerosol barrier (recommended)
• 1.5 mL microcentrifuge tube for lysis
• Microcentrifuge (2 mL tube, rotor-equipped)*
• Ethanol (95–100%), Catalog Nos. E7148, E7023, or 459836
• Molecular Biology Reagent Water, Catalog No. W4502

*Note: To ensure proper fit of all tubes, a 24-place rotor is recommended. If you are using a 36-place rotor, we recommend using every other place for proper tube fit

Storage and Stability

Store the kit at room temperature. If any kit reagent forms a precipitate, warm at 55–65 °C until the precipitate dissolves and allow to cool to room temperature before use.

Preparation Instructions

1. Preheat a Water Bath or Heating Block to 55 °C
2. Thoroughly Mix Reagents
   Examine reagents for precipitation. If any reagent forms a precipitate, warm at 55–65 °C until the precipitate dissolves and allow cooling to room temperature before use.
3. Dilute Prewash Solution
   Concentrate Dilute the concentrate with 5.5 mL (10 prep package), 27.5 mL (70 prep package), or 110 mL (350 prep package) of 95–100% ethanol. After each use, tightly cap the diluted Prewash Solution to prevent the evaporation of ethanol.
4. Dilute Wash Solution Concentrate
   Dilute the concentrate with 10 mL (10 prep package), 80 mL (70 prep package), or 360 mL (350 prep package) of 95–100% ethanol. After each use, tightly cap the diluted Wash Solution to prevent the evaporation of ethanol.
5. Dissolve the Proteinase K
   Dissolve the powder in one bottle of Proteinase K in water to obtain a 20 mg/mL stock solution, according to Table 1. The Proteinase K solution can be stored for several days at 2–8 °C. For longer-term storage, the unused portion of the solution may be stored in aliquots at –20 °C until needed. This product as supplied is stable at room temperature.
   
   Note: The Proteinase K solution must be added directly to each sample every time. Do not combine the Proteinase K and Lysis Solutions for storage.

Catalog No.	Proteinase K (mg)	Water (mL)
NA2010	10	0.5
NA2020	100	5.0

Procedure

See Appendix 1 convert g-force to RPM

Note: If minimally sheared genomic DNA is desired in downstream applications, e.g., if using the end product for long amplification PCR, mix with gentle pipetting or inversion until homogeneous instead of vortexing in the procedure that follows.

1. Collect Blood
   Collect whole blood in an anticoagulant tube (an EDTA tube is preferred). Whole blood should be equilibrated to room temperature before beginning preparation.
   
   
2. Prepare Blood
   Place 20 µL of the Proteinase K solution into a 1.5 mL microcentrifuge tube. Add up to 200 µL of the whole blood sample to the tube. For larger volumes, see Appendix. If the sample is less than 200 µL, add the Resuspension Solution to bring the volume up to 200 µL.
   
   Note: If the sample is already dispensed into a tube, the Proteinase K solution can be added to the sample. Vortex to ensure thorough mixing of the enzyme. Whole blood may be stored at 4 °C for at least 3 months before preparing the DNA. If residual RNA is not a concern, continue with step 3.
   
   Optional RNase A treatment: If RNA-free genomic DNA is required, add 20 µL of RNase A Solution and incubate for 2 minutes at room temperature; continue with step 3.
   
   
3. Lyse Cells
   Add 200 µL of Lysis Solution C to the sample; vortex thoroughly (15 seconds). A homogeneous mixture is essential for efficient lysis. Incubate at 55 °C for 10 minutes.
   
   
4. Column Preparation
   Add 500 µL of the Column Preparation Solution to each pre-assembled GenElute™ Miniprep Binding Column (with a red o-ring, not to be confused with other GenElute™ kits) and centrifuge at 12,000 x g for 1 minute. Discard the flow-through liquid.
   
   Note: The Column Preparation Solution maximizes binding of DNA to the membrane resulting in more consistent yields.
   
   
5. Prepare for Binding
   Add 200 µL of ethanol (95–100%) to the lysate from step 3; mix thoroughly by vortexing for 5–10 seconds. A homogeneous solution is essential.
   
   
6. Load Lysate
   Transfer the entire contents of the tube into the treated column from step 4. Use a wide bore pipette tip to reduce shearing of the DNA when transferring contents into the column. Centrifuge at ≥6500 x g for 1 minute. Discard the collection tube containing the flow-through liquid and place the column in a new 2 mL collection tube.
   
   
7. First Wash
   Prior to first use, dilute both the Prewash Solution Concentrate and the Wash Solution Concentrate with ethanol as described in the Preparation Instructions. Add 500 µL of either Prewash Solution or Wash Solution to the column and centrifuge for 1 minute at ≥6500 x g. Discard the collection tube containing the flow-through liquid and place the column in a new 2 mL collection tube.
   
   Note: If the whole blood sample is aged (older than 24 hours), the Prewash Solution is helpful in removing contaminants associated with older whole blood samples. If the sample is fresh, the Prewash Solution is not always necessary for the first wash.
   
   
8. Second Wash
   Add 500 µL of Wash Solution to the column; centrifuge for 3 minutes at maximum speed (12,000–16,000 x g) to dry the column. The column must be free of ethanol before eluting the DNA. Centrifuge the column for 1 additional minute at maximum speed if residual ethanol is observed. You may empty and re-use the collection tube if you need this additional centrifugation step. Discard the collection tube containing the flow-through liquid and place the column in a new 2 mL collection tube.
   
   
9. Elute DNA
   Pipette 200 µL of the Elution Solution directly into the center of the column. Centrifuge for 1 minute at ≥6500 x g to elute the DNA. To increase the elution efficiency, incubate for 5 minutes at room temperature after adding the Elution Solution, then centrifuge.
   
   Optional: A second eluate can be collected by repeating step 9 with an additional 200 µL of Elution Solution and eluting in a new 2 mL collection tube (not provided) or into the same 2 mL collection tube as used for the first eluate. The eluate contains pure genomic DNA. For short term storage of the DNA, 2–8 °C is recommended. For longer-term storage, –20 °C is recommended. Avoid freezing and thawing, which causes breaks in the DNA strand. The Elution Solution will help stabilize the DNA at these temperatures.

DNA Precipitation (Optional)

The GenElute™ Blood Genomic DNA Kit is designed so that the DNA always remains in solution, which avoids resuspension issues. However, if it is necessary to concentrate the DNA, ethanol precipitation in the presence of sodium acetate is recommended.¹

Results

The concentration and quality of the genomic DNA can be determined by spectrophotometric analysis and agarose gel electrophoresis. Dilute the DNA in TE Buffer (10 mM Tris-HCl, 1 mM EDTA, pH 8.0–8.5) and measure the absorbance at 260 nm, 280 nm, and 320 nm using a quartz microcuvette. The absorbance at 260 nm should be between 0.1 and 1.0 (or within the linear range of your spectrophotometer). The 320 nm absorbance is used to correct for background absorbance. An absorbance of 1.0 at 260 nm corresponds to approximately 50 µg/mL of doublestranded DNA. The A₂₆₀–A₃₂₀/A₂₈₀–A₃₂₀ ratio should be 1.6–1.9.

The size and quality of the DNA can be determined by agarose gel electrophoresis.¹ A gel containing 0.8% agarose (A9539) in 0.55 TBE Buffer (T6400) works well for the resolution of genomic DNA. The DNA can be visualized by staining with an intercalating dye such as ethidium bromide (E1510) and measured against a known DNA marker such as Lambda DNA Hind III digest (D9780). The genomic DNA should migrate as a single, high molecular weight band with very little evidence of shearing. A more precise determination of the size of the DNA can be made by pulsed-field gel electrophoresis.²

Figure 1. Genomic DNA purified by GenElute™ Blood Genomic DNA kits is suitable for restriction enzyme digestions.

Restriction Enzymes, EcoR I and Hind III were used to digest genomic DNA isolated with GenElute™ Blood Genomic DNA kit. Whole blood was collected in 3 different anticoagulants: EDTA, heparin, and sodium citrate. A 100 ng aliquot of genomic DNA from each anticoagulant was initially digested with EcoR I (5 units per 1 μL digested at 37 °C for 1.5 hours) and Hind III (10 units per 1 μL digested at 37 °C for 1.5 hours) followed by electrophoresis (50 ng/lane) on a 0.8% agarose gel. Ladder (M) used was Lambda Hind III (Prod. No. D9780).

1. Sambrook H. 1989. Molecular cloning: a laboratory manual.. Cold Spring Harbor, NY.. https://ci.nii.ac.jp/naid/10003540621/

2. BIRREN B, LAI E. 1993. FIELD INVERSION GEL ELECTROPHORESIS.121-128. https://doi.org/10.1016/b978-0-12-101290-8.50011-0

Troubleshooting Guide

Binding column is clogged.	Cause — Sample is too large. Solution — In the future, use a smaller quantity of whole blood. To salvage the current preparation, increase the g-force and/or spin longer until the lysate passes through the binding column. The yield of genomic DNA may be reduced.
Poor or low genomic DNA recovery.	Cause — Sample is old or degraded. Solution — The yield will vary among individual samples of fresh or aged (older than 24 hours) whole blood. Use whole blood within a few hours of collection for best results. If samples are being stored for future use, whole blood may be kept at 4 °C for at least 3 months.
	Cause — Lysate/ethanol mixture is not homogeneous. Solution — To ensure a homogeneous Solution, vortex 5–10 seconds before applying to the binding column. If minimally sheared genomic DNA is desired in downstream applications, e.g., if using the end product for long amplification PCR, mix with gentle pipetting or inversion until homogeneous instead of vortexing.
	Cause — DNA elution is incomplete. Solution — Confirm that the DNA was eluted in 200 µL of Elution Solution. The DNA yield for most types of material may be improved by incubating the Elution Solution for 5 minutes at room temperature after it is added to the column. A second and third elution using 200 µL of Elution Solution may also be performed.
	Cause — Ethanol was omitted during binding. Solution — Check that the ethanol was added in step 5 before applying the sample to the binding column in step 6.
	Cause — The eluate contains residual ethanol from wash. Solution — Ethanol from the final wash must be eliminated before eluting the DNA. Spin longer or a second time to dry the membrane. If flow-through liquid containing ethanol contacts the binding column, repeat the centrifugation step before eluting DNA.
	Cause — Prewash and/or Wash Solution Concentrates were not diluted before use. Solution — Check that Prewash and Wash Solution Concentrates were properly diluted with ethanol before use.
	Cause — Water was used for elution instead of Elution Solution. Solution — Elution Solution is recommended for optimal yields and storage of the purified DNA. If water is used to elute the DNA, confirm that the pH is at least 7.0, to avoid acidic conditions which may subject the DNA to acid hydrolysis when stored for long periods of time.
Purity of the DNA is lower than expected (A260/A280 ratio is too low).	Cause — Eluate was diluted in water for absorbance measurement.  Solution — Use either Elution Solution (10 mM Tris-HCl, 0.5 mM EDTA, pH 9.0) or 10 mM Tris-HCl, pH 8.0–8.5, as the diluent.
	Cause — Blood sample was older than 24 hours. Solution — In future preparations use the Prewash Solution in the first wash step.
	Cause — Background reading is high due to silica fines. Solution — Spin the DNA sample at maximum speed for 1 minute; use the supernatant to repeat absorbance readings.
	Cause — Purification is incomplete due to column overloading or inadequate lysis. Solution — Reduce the initial volume of blood or increase the lysis time (step 3) while monitoring the lysis visually.
Purity of the DNA is lower than expected (A260/A280 ratio is too high).	Cause — Genomic DNA is contaminated with RNA. Solution — Include an RNase A treatment step in future isolations or treat final product with RNase A Solution and repurify.
DNA is sheared.	Cause — Genomic DNA was handled improperly. Solution — This kit is designed to eliminate DNA precipitation and resuspension, common steps found in other genomic DNA kits that can lead to shearing. All pipetting steps should be executed as gently as possible. Wide-orifice pipette tips are recommended to help eliminate shearing. If minimally sheared genomic DNA is desired in downstream applications, e.g., if using the end product for long amplification PCR, mix with gentle pipetting or inversion until homogeneous instead of vortexing.
	Cause — Blood sample is old, degraded, or has undergone repeated freeze/thaw cycles. Solution — Old starting material may yield degraded DNA in the eluate. For best results, fresh whole blood preparations should be used immediately. Alternatively, whole blood can be stored at 4 °C for up to 3 months.
Downstream applications are inhibited.	Cause — Ethanol is carried over into the final genomic DNA preparation. Solution — After the final wash of the binding column (step 8), do not allow the flow-through liquid to contact the column. Re-spin the column for 1 additional minute at maximum speed (12,000–16,000 x g), if necessary, after emptying the collection tube.
	Cause — Salt is carried over into the final genomic DNA preparation. Solution — Make sure that the binding column is transferred to a new 2 mL collection tube before adding the Prewash or Wash Solutions in step 7.

Appendix 1

Note: All centrifugation speeds are given in units of g. Please refer to Table 2 for information on converting g-force to rpm. If centrifuges/rotors for the required g-forces are not available, use the maximum g-force possible and increase the spin time proportionally. Spin until all liquid passes through the column.

Table 2. Conversion of Centrifugal Force (in units of g) to RPM for Common Rotors

Centrifuge	Rotor	Tubes (max)	Radius (cm)	rpm at 300 x g	rpm at 6,500 x g	rpm at 12,000 x g	rpm at 16,000 x g
Eppendorf 5410	-	12	5.8	2,143	10,012	13,555	15,652
5415C	F45-18-11	18	7.3	1,917	8,924	12,124	14,000
5415D&R	F45-24-11	24	8.3	1,801	8,369	11,392	13,155
5417C,D,&R	F45-30-11	30	9.5	1,681	7,823	10,634	12,279

See table above for spin speeds in RPM for selected common centrifuges and rotors. The correct RPM for unlisted rotorscan be calculated using the formula:

RPM = √[RCF/(r × 1.118)] × 1 × 10⁵

where RCF = required gravitational acceleration (relative centrifugal force) in units of g;
r = radius of the rotor in cm;
rpm = the number of revolutions per minute required to achieve the necessary g-force

Appendix 2: Larger Volumes of Blood

Up to 500 µL of blood can be used with this kit. Reagents must be increased accordingly. (Lysis Solution C and ethanol additions should be approximately 50 µL over the volume of whole blood added in the preparation.) This will decrease the number of preparations you can get from the kit. The binding column will have to be filled and spun several times to load all of the lysate from step 5, depending upon the volume used. For example, add 50 µL of Proteinase K to a 2 mL tube, 500 µL of whole blood, 40 µL of RNase A, and 550 µL of Lysis Solution C. Mix and incubate at 55 °C for 10 minutes. Add 550 µL of 95–100% ethanol and mix. Load onto the binding column (3 x 600 µL) and spin as in step 6. Continue with steps 7–9 as noted in the Procedure.

Precautions and Disclaimer

The GenElute™ Blood Genomic DNA Kit is for laboratory use only, not for drug, household, or other uses.

The Lysis Solution C contains a chaotropic salt, which is an irritant. The Column Preparation Solution is an irritant. Avoid contact with skin. Wear gloves, safety glasses, and suitable protective clothing when handling these solutions or any reagent provided with the kit. Please consult the Safety Data Sheet (SDS) for information regarding hazards and safe handling practices.