CellRapeutics™ GenElute Gel Extraction Kit(CART-015CL)


Product Name
CellRapeutics™ GenElute Gel Extraction Kit
Cat. No.
CART-015CL
General Description
The GenElute Gel Extraction Kit is designed for the rapid purification of 50 bp to 10 Kb linear DNA fragments and plasmids from standard or low-melting agarose gels. This kit can also be used to purify DNA from polyacrylamide gels.
The GenElute Gel Extraction Kit combines silicabinding technology with the convenience of a spin or vacuum column format. DNA fragments of interest are extracted from slices of an agarose gel by solubilizing the gel. The Gel Solubilization Solution can dissolve an agarose slice from gels run in either TBE or TAE buffer. This solution also contains a pH indicator that allows the gel slice to be visualized easily and indicates whether the pH is optimal for DNA binding. The extracted DNA fragments are then selectively adsorbed onto a silica membrane in the presence of the Gel Solubilization Solution. Contaminants are removed by a simple spin or vacuum wash. Finally, the bound DNA is eluted in Tris buffer. The isolated DNA is suitable for a variety of downstream applications, such as automated DNA sequencing, PCR, restriction digestion, cloning, and labeling.
A typical recovery is 50-55%, with recoveries as high as 80%. Each column can bind up to 10 mg of DNA, and up to 3.5 g of agarose can be processed per column.
Components
Sufficient for 70 preparations
Column Preparation Solution 60 ml
Gel Solubilization Solution 140 ml
Wash Solution Concentrate G 12 ml
Elution Solution (10 mM Tris-HCl, pH 9.0) 6 ml
GenElute Binding Column G 70 each
Collection Tubes, 2 ml 2 X 70 each
Storage Conditions
Store the kit at room temperature
Reagent and instrument requirements
Equipment and Reagents Required But Not Provided · Cutting tools for gel or razor blades
· Pipettors and tips
· Water bath or heating block at 50-60 °C
· Ethanol, 95-100%
· Isopropanol, 99-100%
· Microcentrifuge and tubes
· Water, Molecular Biology Reagent
· 3 M Sodium Acetate Buffer, pH 5.2
Procedure
Preparation Instructions: 1. Wash Solution: Dilute the entire 12 ml of the Wash Solution Concentrate G with 48 ml of 95-100% ethanol prior to initial use. After each use, tightly cap the diluted Wash Solution to prevent the evaporation of ethanol. 2. Gel Solubilization Solution: The Gel Solubilization Solution will precipitate out of solution if stored at temperatures less than 18-25 °C. Check to ensure that this solution is completely dissolved and that no crystals are present. If crystals are present, incubate at 37-50 °C with periodic mixing until the crystals dissolve (approximately 5 minutes). 3. Agarose Gel Electrophoresis: Use fresh electrophoresis running buffer. Electrophoresis buffer, which has been used repeatedly, will reduce the DNA recovery efficiency. Minimize examination of ethidium bromide-stained gels with an UV transilluminator. If possible, use a transilluminator equipped with a long-wavelength (302 nm) UV light source, as this will minimize the damaging effects of UV light on nucleic acids. 4. Elution Solution: If purifying plasmid DNA or large linear DNA fragments (>3 Kb) preheat the Elution Solution to 65 °C prior to use. A. Spin Procedure for Agarose Gels All centrifugations (spins) are performed at 12,000 to 16,000 x g. 1a. Excise band. Excise the DNA fragment of interest from the agarose gel with a clean, sharp scalpel or razor blade. Trim away excess gel to minimize the amount of agarose.
2a. Weigh gel. Weigh the gel slice in a tared colorless tube.
3a. Solubilize gel. Add 3 gel volumes of the Gel Solubilization Solution to the gel slice. In other words, for every 100 mg of agarose gel, add 300 mL of Gel Solubilization Solution. Incubate the gel mixture at 50-60 °C for 10 minutes, or until the gel slice is completely dissolved. Vortex briefly every 2-3 minutes during incubation to help dissolve the gel.
Note: To adequately dissolve a gel with an agarose concentration greater than 2%, it is necessary to increase the ratio of the Gel Solubilization Solution volume to the gel weight to 6:1.
4a. Prepare binding column. Preparation of the binding column can be completed while the agarose is being solubilized in step 3a. Place the GenElute Binding Column G into one of the provided 2 ml collection tubes. Add 500 mL of the Column Preparation Solution to each binding column. Centrifuge for 1 minute. Discard flowthrough liquid. Note: The Column Preparation Solution maximizes binding of DNA to the membrane resulting in more consistent yields.
5a. Check the color of the mixture. Once the gel slice is completely dissolved (step 3a) make sure the color of the mixture is yellow (similar to fresh Gel Solubilization Solution with no gel slice) prior to proceeding to the following step. If the color of the mixture is red, add 10 mL of the 3 M Sodium Acetate Buffer, pH 5.2, and mix. The color should now be yellow. If not, add the 3 M Sodium Acetate Buffer, pH 5.2, in 10 mL increments until the mixture is yellow.
6a. Add isopropanol. Add 1 gel volume of 100% isopropanol and mix until homogenous. For a gel with an agarose concentration greater than 2%, use 2 gel volumes of 100% isopropanol.
7a. Bind DNA. Load the solubilized gel solution mixture from step 6a into the binding column that is assembled in a 2 ml collection tube. It is normal to see an occasional color change from yellow to red once the sample is applied to the binding column. If the volume of the gel mixture is>700 mL, load the sample onto the column in 700 mL portions. Centrifuge for 1 minute after loading the column each time. Discard the flowthrough liquid. Note: Do not be alarmed if the flow-through has changed color.
8a. Wash column. Add 700 mL of Wash Solution (diluted from Wash Solution Concentrate G as described under Preparation Instructions) to the binding column. Centrifuge for 1 minute. Remove the binding column from the collection tube and discard the flow-through liquid. Place the binding column back into the collection tube and centrifuge again for 1 minute without any additional wash solution to remove excess ethanol. Residual Wash Solution will not be completely removed unless the flow-through is discarded before the final centrifugation. 9a. Elute DNA. Transfer the binding column to a fresh collection tube. Add 50 mL of Elution Solution to the center of the membrane and incubate for 1 minute. Centrifuge for 1 minute. For efficient recovery of intact plasmid DNA, preheat the elution solution to 65 °C prior to adding it to the membrane. Eluting at 65 °C improves plasmid DNA recoveries by 2 to 3-fold. Yields of large linear DNA fragments (>3 Kb) can also be increased by up to 20% by preheating the elution solution to 65 °C.
Note: To increase the concentration of the eluted DNA, the volume of Elution Solution may be reduced to 25 mL. Yields are ~25% lower when eluting with 25 mL as opposed to 50 mL. B. Vacuum Procedure for Agarose Gels The GenElute Miniprep Binding Columns are designed for use with any vacuum manifold with luer connectors.
1b. Excise band. Excise the DNA fragment of interest from the agarose gel with a clean, sharp scalpel or razor blade. Trim away excess gel to minimize the amount of agarose.
2b. Weigh gel. Weigh the gel slice in a tared colorless tube.
3b. Solubilize gel. Add 3 gel volumes of the Gel Solubilization Solution to the gel slice. In other words, for every 100 mg of agarose gel, add 300 mL of Gel Solubilization Solution. Incubate the gel mixture at 50-60 °C for 10 minutes, or until the gel slice is completely dissolved. Vortex briefly every 2-3 minutes during incubation to help dissolve the gel.
Note: To adequately dissolve a gel with an agarose concentration greater than 2%, it is necessary to increase the ratio of the Gel Solubilization Solution volume to the gel weight to 6:1.
4b. Prepare binding column. Preparation of the binding column can be completed while the agarose is being solubilized in step 3b. Place the GenElute Binding Column G onto the vacuum manifold. Apply vacuum and add 500 mL of the Column Preparation solution to the column. Allow the Column Preparation Solution to pass completely though the column.
Note: The Column Preparation Solution maximizes binding of DNA to the membrane resulting in more consistent yields. 5b. Check the color of the mixture. Once the gel slice is completely dissolved (step 3b) make sure the color of the mixture is yellow (similar to fresh Gel Solubilization Solution with no gel slice) prior to proceeding to the following step. If the color of the mixture is red, add 10 mL of 3 M Sodium Acetate, pH 5.2, and mix. The color should now be yellow. If not, add the 3 M Sodium Acetate Buffer, pH 5.2, in 10 mL increments until the mixture is yellow.
6b. Add isopropanol. Add 1 gel volume of 100% isopropanol and mix briefly until homogenous. For a gel with an agarose concentration greater than 2%, use 2 gel volumes of 100% isopropanol.
7b. Bind DNA. With vacuum applied, load the solubilized gel solution mixture from step 6b into the binding column that is attached to the vacuum manifold. Be careful not to overfill. Allow the mixture to pass through the column. It is normal to see an occasional color change from yellow to red once the sample is applied to the column. Do not be alarmed if the flow-through has changed color.
8b. Wash column. Add 700 mL of Wash Solution (diluted from Wash Solution Concentrate G as described under Preparation Instructions) to the binding column and allow it to pass through.
9b. Transfer column. Remove the binding column from the vacuum manifold and transfer it to a clean 2 ml collection tube. Centrifuge for 1 minute at 12,000 to 16,000 x g to remove excess ethanol.
10b. Elute DNA. Transfer the binding column to a fresh collection tube. Add 50 mL of Elution Solution to the center of the membrane and incubate for 1 minute. Centrifuge for 1 minute. For efficient recovery of intact plasmid DNA, preheat the elution solution to 65 °C prior to adding it to the membrane. Eluting at 65 °C improves plasmid DNA recoveries by 2 to 3-fold. Yields of large linear DNA fragments (>3 Kb) can also be increased by up to 20% by preheating the elution solution to 65 °C.
Note: To increase the concentration of the eluted DNA, the volume of Elution Solution may be reduced to 25 mL. Yields are ~25% lower when eluting with 25 mL as opposed to 50 mL. C. Extraction of DNA from Polyacrylamide Gels The GenElute Gel Extraction Kit is designed for isolating DNA from agarose gels; however, the kit can also be used to isolate DNA from polyacrylamide gels when using the following "crush and soak" method. The Gel Diffusion Buffer, which is not included in the kit, must be prepared prior to beginning the procedure. All centrifugations (spins) are performed at 12,000 to 16,000 x g. 1c. Prepare Gel Diffusion Buffer. The Gel Diffusion Buffer consists of 0.1% SDS, 1 mM EDTA, 10 mM magnesium acetate, and 500 mM ammonium acetate, pH 8.0. To make 100 ml, add 50 ml nuclease-free water to an appropriately sized beaker and stir. Add the components in the order that they are listed (see Table 1) while stirring. Continue to mix until all components are thoroughly dissolved. Bring up to 100 ml with nuclease-free water. The ammonium acetate must be added last to avoid precipitation of the SDS. Note: If the SDS precipitates, incubate at 37-50 °C with periodic mixing until the crystals dissolve (approximately 5 minutes). 2c. Excise Band. Excise the DNA fragment of interest from the polyacrylamide gel with a clean, sharp scalpel or razor blade. Trim away excess gel to minimize the amount of polyacrylamide. Transfer the gel slice into a tared 1.5 ml colorless microcentrifuge tube.
3c. Crush Gel. Crush the gel slice using a clean disposable pipette tip (i.e., a 200 mL pipette tip) or similar device by compressing it between the side of the tube using the pipette tip. The smaller the pieces the better. Breaking the gel slice into tiny pieces results in up to 50% greater yields of DNA due to the increased surface area available for passive diffusion.
Note: If pieces of gel become lodged in the pipette tip, they can easily be removed by briefly centrifuging the pipette tip in the opened microcentrifuge tube. The tip can then be discarded.
4c. Weigh gel. Weigh the gel in the tube in which it is crushed.
5c. Diffuse DNA from gel. Resuspend the gel pieces in 2 gel volumes of Gel Diffusion Buffer. In other words, for every 50 mg of gel, add 100 mL of Gel Diffusion Buffer. Make certain that all pieces are submerged in the Gel Diffusion Buffer. Incubate at 50 °C for a minimum of 30 minutes. For the best yields, the incubation time can be extended overnight.
6c. Remove Residual Polyacrylamide. Spin the crushed gel/buffer mixture at 12,000 to 16,000 x g for 1 minute and carefully remove the supernatant to a new microcentrifuge tube being careful to avoid transferring fragments of polyacrylamide. Alternatively, residual polyacrylamide can be removed by passing the mixture through a disposable plastic column (Catalog No. 56500).
7c. Optional: For optimal yield, add an additional 0.5 gel volume of Gel Diffusion Buffer to the pelleted polyacrylamide. Vortex briefly to resuspend the pellet and repeat steps 5c and 6c. Pool the supernatant and proceed to the next step.
8c. Measure the volume of recovered supernatant and add 3 volumes of the Gel Solubilization Solution. For every 100 mL of supernatant, add 300 mL of Gel Solubilization Solution.
9c. Continue with step 4 of either the Vacuum or Spin Procedure.
Product Use Statement
The GenElute Gel Extraction Kit is for laboratory use only and is not intended for drug, household, or other uses. The Gel Solubilization Solution contains a chaotropic salt, which is an irritant. The Column Preparation Solution is also an irritant. Wear gloves, safety glasses, and suitable protective clothing when handling this solution or any reagents provided with the kit.

For research use only. Not intended for any clinical use. No products from Creative Biolabs may be resold, modified for resale or used to manufacture commercial products without prior written approval from Creative Biolabs.

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