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Polyacryl Gel DNA Purification Kit: Purification of DNA fragment from polyacryl gel.

Introduction

The EZgeneTM Poly-Gel DNA Extraction Kit combines the simplicity of PAGE with the power of ezBind technology to allow rapid and consistent recovery of ssDNA or dsDNA from acrylamide gels. Following PAGE, oligonucleotides are first visualized by UV shadowing or ethidium bromide staining and then eluted in 1-4 h using EDTA-containing buffer to eliminate degradation of DNA by contaminating DNase. A specially formulated buffer is then added to the eluate which is passed through an ezBind DNA spin column. The bound DNA is finally washed to remove salts, buffers, and any protein contaminants, and then eluted with sterile deionized water or low salt buffer. Organic extractions and alcohol precipitations are eliminated reducing sample handling time and increasing yields. Furthermore, the spin column format allows parallel processing of multiple samples. The purified DNA is suitable for PCR, ligation reactions, hybridization techniques, or other procedures.

New in This Edition

A new column Equilibrate Buffer is introduced in this kit to improve the DNA yield and kit performance.

Storage and Stability

All components of the EZgeneTM Poly-Gel DNA Extraction Kit are stable for at least 24 months from date of purchase when stored at 22 ℃-25 ℃. Under cool ambient conditions (such as occurs during shipping) a precipitate may form in buffers, simply warm up the solution to dissolve the precipitates.

Polyacrylamide gel DNA purification protocol

Poly Gel DNA Purification Cat#: DC3512-01 Size: 50 Purification of DNA fragment from polyacryl gel Learn More $ 50.00 Add to Wishlist Add to Compare

Poly Gel DNA Purification Cat#: DC3512-02 Size: 250 Purification of DNA fragment from polyacryl gel Learn More $ 220.00 Add to Wishlist Add to Compare

Acrylamide gels are useful for separation of small DNA fragments, typically oligonucleotides<100 base pairs. These gels are usually of a low acrylamide concentration (≤6%) and contain the non-ionic denaturing agent urea (7 M). For gel composition and buffers for your particular requirements, please refer to the Appendix.

Visualization of DNA

Fluorescence Shadowing DNA fragments resolved on polyacrylamide gels can be visualized by the method of UV shadowing. This method is straight forward and may gives higher yields compared to staining methods (see DNA Staining below). In this technique the gel is placed on top of a flourescent material, usually a flourescent TLC silica plate. The gel is then illuminated by a UV light source. DNA bands in the gel will block transmittance of the UV light to the substrate. This will result in a dark area (i.e. non-fluorescing) area on the substrate

1. The gel must be removed from glass plates (glass blocks UV light and will prevent visualization by either UV shadowing or staining) and wrapped in plastic wrap to aid in handling and marking. Remove the top glass plate, and lay a sheet of plastic wrap over the gel, then flip the gel & glass plate over and carefully peel the gel away from the bottom glass plate. Wrap the gel entirely in the plastic wrap.

TIP: Use only a single layer of plastic wrap and try to prevent air bubbles from forming between the gel and plastic wrap. These bubbles can scatter the UV light and make visualization difficult

2. Place the gel on top of the dull white side of a fluor-coated TLC plate (Ambion, 10110 or Merck 60F254) and remove the plastic wrap on top

of the gel. Hold a hand-held short-wavelength (254 nm) UV light source over the gel. (Long wavelength UV light will not work). The TLC plate beneath the gel should glow bright purple except wherever nucleic acids are present. A DNA band will appear as a dark shadow. The limit of sensitivity is about 0.3 μg in a single band

TIP: UV shadowing works for either DNA or RNA, labeled or unlabeled, so this technique has many other applications; e.g. for visualizing restriction enzyme digests. DNA Staining As an alternative to UV shadowing the acrylamide gel may be stained with acridine orange or ethidium bromide (EtBr) and held over a UV transilluminator to visualize the location of DNA bands within the gel. If the DNA is used as a probe, it is important that the stain be completely removed before hybridization as it will compromise hybridization efficiency. We recommend staining with acridine orange as opposed to EtBr since acridine orange will be removed from the probe by subsequent purification with ezBind DNA spin columns. (EtBr can be used, but requires multiple butanol extractions to subsequently remove it before applying sample to spin column).

1. Remove gel from glass plates as described for UV shadowing.

2. Remove gel from the plastic wrap and place in a 2.0 μg/mL acridine orange solution for 15 minutes. Destain the gel in distilled water for 10 minutes. Then re-wrap the gel in plastic wrap for easier handling, and place the gel on a UV transilluminator to visualize DNA

3. Carefully cut out (using a nuclease-free scalpel or razor blade) the smallest gel-fragment possible which contains the DNA band (corresponds to bright band in the gel). The smaller the size of this gel

fragment, the better the elution efficiency (i.e. more probe will be recovered more quickly). If you are concerned that not all the probe was cut out, visualize the gel again with UV light to verify that the probe band is gone.

Isotopically Labeled DNA If the DNA band of interest has been labeled with 32P, 33P, or 35S for use as a probe, it can be readily visualized by autoradiography

1. Following PAGE separate the glass plates, leaving the gel adhered to the larger glass plate. Wrap a piece of plastic wrap over the gel. If the glass and gel will not fit into the film cartridge, then both glass plates should be carefully removed and the gel wrapped entirely in plastic wrap (for easier handling).The gel is ready to expose to film

2. Place the gel (sandwiched between the glass and plastic wrap) against the film so that the film is closest to the gel. The film can simply be aligned with one corner of the glass plate, the corners and sides of the glass plate marked directly on the film with a permanent marker, or alternatively, radioactive ink can be used for orientation. One corner of the film (e.g. bottom right corner) is usually snipped or folded up so that the glass and gel can be aligned with the film after developing

3. Expose the gel to autoradiographic film, about 30 seconds for a high specific activity 32P-labeled probe and 10 minutes for a low specific activity P-labeled probe or high specific activity S-labeled probe. The aim is to get an exposure of a light gray band so that a thin gel fragment can be excised from the gel. Realign the glass plate and gel with the developed film (using the guide marks made earlier) and carefully excise the band using a nuclease-free scalpel or razor blade. The smaller the size of this gel fragment, the better the elution efficiency (i.e.

more probe will be recovered more quickly). The gel can be re-exposed to insure that the gel and film were properly aligned and that the probe was excised.

TIP: If possible, run markers or a known size standard so that the appropriate band is selected. If no markers have been run, the bromophenol (dark blue) and xylene cyanol (light blue) dyes can serve as size references. See Appendix for dye migration under different conditions.

Poly-Gel DNA Extraction Procedure Please take a few minutes to read and familiarize yourself with this procedure. Make sure all necessary reagents and equipment are ready before starting. Wash Buffer Concentrate must be diluted with absolute ethanol as indicated in Before Starting on page 3 and stored at room temperature.

1. Transfer the gel fragment onto a nuclease-free microscope slide. With a second glass slide (or nuclease-free razor) mash and pulp the gel completely. Carefully transfer gel pulp to a nuclease-free microcentrifuge tube and add 250 μL Gel Elution Buffer. This volume is usually enough to submerge a slice 2 mm x 10 mm x 0.8 mm. For a larger fragment adjust volume of Elution Buffer used until the gel is covered. Any buffer or ddH2O can be used; however, we recommend the Elution Buffer supplied in order to prevent DNA degradation by exogenous nucleases

2. Incubate the gel fragment in Elution Buffer 1-4 h at 65 ℃.The elution time is dependent on the size of the gel fragment, DNA size and the temperature of the incubation. We find that about 70% of a 100 bp DNA fragment elutes in approximately 4 hrs at 65 ℃. Larger fragments will take longer to elute.

3. Transfer Gel and Buffer to a White Poly-Gel Filter Unit mounted in a sterile 1.5 mL microcentrifuge tube. Use a blue pipette tip with the end cut to do this. Centrifuge at 10,000 x g for 10 minutes at room temperature to filter the eluted DNA.