Alkaline Agarose Gel Electr...

Alkaline Agarose Gel Electrophoresis -- Sambrook and Russell 2006 (2... 

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Please cite as: CSH Protocols; 2006; doi:10.1101/pdb.prot4027 

Protocol 

Alkaline Agarose Gel Electrophoresis 

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 

Alkaline agarose gels are run at a pH that is sufficiently high to denature double-stranded DNA. The 

denatured DNA is maintained in a single-stranded state and migrates through the alkaline gel as a function 

of its size. Alkaline agarose gels are used chiefly to measure the size of first and second strands of cDNA 

(Construction of cDNA Libraries Stage 1: Synthesis of First-strand cDNA Catalyzed by Reverse 

Transcriptase) and to analyze the size of the DNA strand after digestion of DNA-RNA hybrids with 

nucleases such as S1. 

MATERIALS 

10x Alkaline agarose gel electrophoresis buffer 

1x TAE electrophoresis buffer 

6x Alkaline gel-loading buffer 

Agarose 

DNA samples (usually radiolabeled) 

DNA staining solution 

For a discussion of staining agarose gels, please see Detection of DNA in Agarose Gels. 

Ethanol 

Optional, please see Step 5. 

Neutralizing solution for alkaline agarose gels 

Sodium acetate (3 M, pH 5.2) 

METHOD 

1. Prepare the agarose solution by adding the appropriate amount of powdered agarose (please see



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Agarose Gel Electrophoresis) to a measured quantity of H 2 O in an Erlenmeyer flask or a glass 

bottle. 

2. Loosely plug the neck of the Erlenmeyer flask with Kimwipes. When using a glass bottle, make 

sure that the cap is loose. Heat the slurry in a boiling-water bath or a microwave oven until the 

agarose dissolves. 

Heat the slurry for the minimum time required to allow all of the grains of agarose to dissolve. 

Check that the volume of the solution has not been decreased by evaporation during boiling; 

replenish with H 2 O if necessary. 

3. Cool the clear solution to 55'C. Add 0.1 volume of 10x alkaline agarose gel electrophoresis 

buffer, and immediately pour the gel as described in Agarose Gel Electrophoresis. After the gel is 

completely set, mount it in the electrophoresis tank and add freshly made 1x alkaline electrophoresis 

buffer until the gel is just covered. 

Do not add ethidium bromide because the dye will not bind to DNA at high pH. 

The addition of NaOH to a hot agarose solution causes hydrolysis of the polysaccharide. For this 

reason, the agarose is first melted in H 2 O and then made alkaline by the addition of NaOH just 

before the gel is poured. 

4. Collect the DNA samples by standard precipitation with ethanol. Dissolve the damp precipitates 

of DNA in 10-20 µl of 1x gel buffer. Add 0.2 volume of 6x alkaline gel-loading buffer. 

It is important to chelate all Mg 2+ with EDTA before adjusting the electrophoresis samples to 

alkaline conditions. In solutions of high pH, Mg 2+ forms insoluble Mg(OH) 2 precipitates that 

entrap DNA. 

It is not strictly necessary to denature the DNA with base before electrophoresis. The exposure of 

the samples to the alkaline conditions in the gel is usually enough to render the DNA singlestranded. 

5. Load the DNA samples dissolved in 6x alkaline gel-loading buffer into the wells of the gel as 

described in Agarose Gel Electrophoresis. Start the electrophoresis at



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DNA to an uncharged nitrocellulose or nylon membrane as described in Southern Blotting: 

Capillary Transfer of DNA to Membranes. 

Alternatively, transfer the DNA directly (without soaking the gel) from the alkaline agarose 

gel to a charged nylon membrane (please see Southern Blotting: Capillary Transfer of DNA 

to Membranes). 

Detect the target sequences in the immobilized DNA by hybridization to an appropriate 

labeled probe (please see Southern Hybridization of Radiolabeled Probes to Nucleic Acids 

Immobilized on Membranes). 

Staining 

Soak the gel in neutralizing solution for 45 minutes at room temperature. 

Stain the neutralized gel with 0.5 µg/ml ethidium bromide in 1x TAE or with SYBR Gold. 

A band of interest can be sliced from the gel and subsequently eluted by one of the 

procedures described in Recovery of DNA from Agarose Gels: Electrophoresis onto 

DEAE-cellulose Membranesor Recovery of DNA from Agarose and Polyacrylamide Gels: 

Electroelution into Dialysis Bags. 

REFERENCES 

1. McDonell, M.W., Simon, M.N., and Studier, F.W. 1977. Analysis of restriction fragments of T7 DNA 

and determination of molecular weights by electrophoresis in neutral and alkaline gels. J. Mol. Biol. 110: 

119–146.[Medline] 

Caution 

Sodium acetate (NaOAc) 

Sodium acetate (NaOAc), see Acetic acid 

Recipe 

Alkaline Gel-loading Buffer 

300 mM NaOH 

6 mM EDTA 

18% (w/v) Ficoll (Type 400, Pharmacia) 

0.15% (w/v) bromocresol green 

0.25% (w/v) xylene cyanol



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For a 6x buffer. 

Recipe 

Alkaline agarose gel electrophoresis buffer 

For 10x solution: Add 50 ml of 10 N NaOH and 20 ml of 0.5 M EDTA (pH 8.0) to 800 ml of H 2 O and 

then adjust the final volume to 1 liter. Dilute the 10x alkaline agarose gel electrophoresis buffer with H 2 O 

to generate a 1x working solution immediately before use. Use the same stock of 10x alkaline agarose gel 

electrophoresis buffer to prepare the alkaline agarose gel and the 1x working solution of alkaline 

electrophoresis buffer. 

Recipe 

DNA staining solution 

Ethidium bromide (10 mg/ml) 

Alternatively, SYBR Gold may be used. 

or SYBR Gold solution 

Recipe 

Neutralizing Solution for Alkaline Agarose Gels 

1 M Tris-Cl (pH 7.6) 

1.5 M NaCl 

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.



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Recipe 

TAE 

Prepare a 50X stock solution in 1 L of H 2 O: 

242 g of Tris base 

57.1 mL of glacial acetic acid 

100 mL of 0.5 M EDTA (pH 8.0) 

The 1X working solution is 40 mM Tris-acetate/1 mM EDTA. 

Copyright © 2006 by Cold Spring Harbor Laboratory Press. Online ISSN: 1559-6095 Terms of Service All 

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