Please cite as: CSH Protocols; 2006; doi:10.1101/pdb.prot2936 Protocol Isolation of DNA Fragments from Polyacrylamide Gels by the Crush and Soak Method Joseph Sambrook and David W. Russell This protocol was adapted from Molecular Cloning, 3rd edition, by Joseph Sambrook and David W. Russell. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, USA, 2001 INTRODUCTION The "crush and soak" method, which works best for DNAs <1 kb in size, can be used to recover both singleand double-stranded DNAs from polyacrylamide gels. The yield of eluted DNA varies from <30% to >90% depending on the size of the DNA fragment. MATERIALS 6x Gel-loading buffer Acrylamide gel elution buffer Chloroform DNA markers generated by restriction digests of known quantities of the DNA sample DNA sample Ethanol Optional, please see Step 5. Phenol:chloroform (1:1, v/v) Sodium acetate (3 M, ph 5.2) TE (ph 8.0) METHOD 1. Carry out polyacrylamide gel electrophoresis of the DNA sample and markers as described in Neutral
Polyacrylamide Gel Electrophoresis. Locate the DNA of interest by autoradiography (Detection of DNA in Polyacrylamide Gels by Autoradiography) or by examination of ethidium bromide- or SYBR Gold-stained gels in long-wavelength (302 nm) UV light (Detection of DNA in Polyacrylamide Gels by Staining). 2. Use a clean sharp scalpel or razor blade to cut out the segment of the gel containing the band of interest, keeping the size of the polyacrylamide slice as small as possible. This can be achieved by any of the following methods: While the DNA is illuminated with UV light, cut through both the gel and the Saran Wrap, and then peel the small piece of gel containing the DNA from the Saran Wrap. Use a permanent felt-tipped marker (e.g., Sharpie pen) to outline the DNA band on the back of the glass plate while the gel is illuminated from below with UV light. Invert the gel, remove the Saran Wrap, and cut out the band using the marker outline as a guide. In the case of a fragment of DNA identified by autoradiography, place the exposed autoradiographic film on the Saran Wrap and align it with the gel. Use a permanent marker to outline the position of the desired DNA fragment on the back of the glass plate. Remove the exposed film and Saran Wrap and cut out the band. Photograph or autoradiograph the gel after the bands of DNA have been excised to produce a permanent record of the experiment. 3. Transfer the gel slice to a microcentrifuge tube or a polypropylene tube. Use a disposable pipette tip or inoculating needle to crush the polyacrylamide gel against the wall of the tube. 4. Calculate the approximate volume of the slice and add 1-2 volumes of acrylamide gel elution buffer to the microcentrifuge tube. 5. Close the tube and incubate it at 37 C on a rotating wheel or rotary platform. At this temperature, small fragments of DNA (<500 bp) are eluted in 3-4 hours; larger fragments take 12-16 hours. 6. Centrifuge the sample at maximum speed for 1 minute at 4 C in a microcentrifuge. Transfer the supernatant to a fresh microcentrifuge tube, being extremely careful to avoid transferring fragments of polyacrylamide (a drawn-out Pasteur pipette works well). 7. Add an additional 0.5 volume of acrylamide gel elution buffer to the pellet of polyacrylamide, vortex briefly, and centrifuge again. Combine the supernatants. 8. (Optional) Remove any remaining fragments of polyacrylamide by passing the supernatant through a disposable plastic column equipped with a frit (e.g., Isolabs, Inc., Quick-Sep columns) or a syringe
barrel plugged with a Whatman GF/C filter or siliconized glass wool. The eluted DNA can be extracted with phenol:chloroform and chloroform to remove SDS, which can inhibit subsequent enzymatic manipulation of the DNA. Precipitate the extracted DNA with ethanol as described in Step 9 and continue with the remainder of the protocol. 9. Add 2 volumes of ethanol at 4 C to the flow-through and store the solution on ice for 30 minutes. Recover the DNA by centrifugation at maximum speed for 10 minutes at 4 C in a microcentrifuge. Even small quantities of DNA are efficiently precipitated by ethanol in this method. However, 10 µg of carrier RNA can be added before precipitation, which may improve even further the recovery of small amounts of DNA. Before adding the RNA, make sure that the presence of RNA will not compromise subsequent reactions with the DNA. (For preparation of carrier RNA, please see Recovery of DNA from Agarose Gels: Electrophoresis onto DEAE-cellulose Membranes.) 10. Dissolve the DNA in 200 µl of TE (ph 8.0), add 25 µl of 3 M sodium acetate (ph 5.2), and again precipitate the DNA with 2 volumes of ethanol as described in Step 9. 11. Carefully rinse the pellet once with 70% ethanol, and dissolve the DNA in TE (ph 8.0) to a final volume of 10 µl. 12. Check the amount and quality of the fragment by polyacrylamide or high-resolution agarose gel electrophoresis: Mix a small aliquot (approx. 20 ng) of the final preparation of the fragment with 10 µl of TE (ph 8.0), and add 2 µl of the desired gel-loading buffer. Load and run a polyacrylamide or high-resolution agarose gel of the appropriate concentration, using as markers restriction digests of known quantities of the original DNA. The isolated fragment should comigrate with the correct fragment in the restriction digest. Examine the gel carefully for the presence of faint fluorescent bands that signify the presence of contaminating species of DNA. It is often possible to estimate the amount of DNA in the final preparation from the relative intensities of fluorescence of the fragment and the markers. REFERENCES 1. Maxam, A.M. and Gilbert, W. 1980. Sequencing end-labeled DNA with base-specific chemical cleavages. Methods Enzymol. 65: 499 560.[Medline] 2. Maxam, A.M. and Gilbert, W. 1977. A new method for sequencing DNA. Proc. Natl. Acad. Sci. 74: 560 564.[Abstract/Free Full Text]
Caution Chloroform (CHCl 3 ) Chloroform (CHCl 3 ) is irritating to the skin, eyes, mucous membranes, and respiratory tract. It is a carcinogen and may damage the liver and kidneys. It is also volatile. Avoid breathing the vapors. Wear appropriate gloves and safety glasses. Always use in a chemical fume hood. Caution Phenol:chloroform Phenol is extremely toxic, highly corrosive, and can cause severe burns. It may be harmful by inhalation, ingestion, or skin absorption. Wear appropriate gloves, goggles, and protective clothing. Always use in a chemical fume hood. Rinse any areas of skin that come in contact with phenol with a large volume of water and wash with soap and water; do not use ethanol! Chloroform (CHCl 3 ) is irritating to the skin, eyes, mucous membranes, and respiratory tract. It is a carcinogen and may damage the liver and kidneys. It is also volatile. Avoid breathing the vapors. Wear appropriate gloves and safety glasses. Always use in a chemical fume hood. Caution Sodium acetate (NaOAc) Sodium acetate (NaOAc), see Acetic acid Recipe 6x Gel-loading buffer 30% glycerol 0.25% bromophenol blue
Recipe Acrylamide gel elution buffer 10 mm magnesium acetate tetrahydrate 0.5 M ammonium acetate 1 mm EDTA (ph 8.0) 0.1% (w/v) SDS (optional) SDS improves the efficiency of recovery, most probably by blocking nonspecific adsorption of DNA to the walls of the tube. However, SDS is tenacious and difficult to remove from the eluted DNA, especially when purifying oligonucleotides on Sep-Pak columns. Perhaps the best advice is to use SDS only when attempting to recover very small amounts (<20 ng) of DNA >1 kb in size, where recovery is already inefficient and further losses may prejudice the success of the experiment. This is not usually the case when purifying synthetic oligonucleotides, which are always relatively small and usually available in abundance. Other buffers may be substituted for acrylamide gel elution buffer. For example, if the DNA fragment is radiolabeled and is to be used as a hybridization probe, hybridization buffer can be substituted. Recipe Sodium acetate To prepare a 3 M solution: Dissolve 408.3 g of sodium acetate 3H 2 O in 800 ml of H 2 O. Adjust the ph to 5.2 with glacial acetic acid or to 7.0 with dilute acetic acid. Adjust the volume to 1 L with H 2 O. Dispense into aliquots and sterilize by autoclaving. Recipe TE buffer, 10X 100 mm Tris-Cl (desired ph) 10 mm EDTA (ph 8.0) Sterilize solutions by autoclaving for 20 min at 15 psi (1.05 kg/cm 2 ) on liquid cycle. Store the buffer at room
temperature. Copyright 2006 by Cold Spring Harbor Laboratory Press. Online ISSN: 1559-6095 Terms of Service All rights reserved. Anyone using the procedures outlined in these protocols does so at their own risk. Cold Spring Harbor Laboratory makes no representations or warranties with respect to the material set forth in these protocols and has no liability in connection with their use. All materials used in these protocols, but not limited to those highlighted with the Warning icon, may be considered hazardous and should be used with caution. For a full listing of cautions, click here. All rights reserved. No part of these pages, either text or images, may be used for any reason other than personal use. Reproduction, modification, storage in a retrieval system or retransmission, in any form or by any means-electronic, mechanical, or otherwise-for reasons other than personal use is strictly prohibited without prior written permission. CiteULike Connotea Del.icio.us Digg Reddit Technorati What's this?