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A technique for radiolabeling DNA restriction endonuclease fragments to high specific activity
ANALYl
ICAL
BIOCHEMISTRY
137, 266-267
(I 984)
ADDENDUM “A Technique
for Radiolabeling DNA Restriction Endonuclease Fragments to High Specific Activity” Volume
132. Number
1 (1983),
Volume 132, Number 1 (1983) in the article “A Technique for Radiolabeling DNA Restriction Endonuclease Fragments to High Specific Activity.” by Andrew P. Feinberg and Bert Vogelstein, pages 6- 13, we described a method for recovering DNA restriction endonuclease fragments from agarose gels and labeling them to high specific activity. We have since found that a modification of the labeling technique can be used to label DNA restriction fragments without purifying the DNA from the agarose. The modified technique described below eliminates the loss of DNA and time involved in recovering DNA fragments from agarose gels and can be used even with very small amounts of template DNA. (1) Plasmid or phage DNA is cleaved with an appropriate restriction endonuclease and the fragments are electrophoretically separated in one lane of a l-2% low gelling temperature agarose gel (Bio-Rad No. 162-00 17). The electrophoresis buffer is 40 mM Tris-acetate, 5 mM sodium acetate, 1 mM EDTA at pH 8.0. Generally, 4-8 hg of DNA can be run in a 24-mm’ lane without loss of resolution. After electrophoresis, the gel (10 X 14 X 0.3 cm) is stained for 20-30 min in a liter of HZ0 containing 0.2 pg/ml of ethidium bromide. (2) The desired band is excised cleanly and put into a preweighed 1.5-ml microcentrifuge tube. HZ0 is added at a ratio of 3 ml H20/g of gel. The tube is then placed in a boiling water bath for 7 min to dissolve the gel and denature the DNA. It is stored at -20°C. Pre-
0003-2697/84
$3.00
266
Pages 6-13
paratory to subsequent labelings, the gel is reboiled for 3 min and briefly stored (lo-60 min) in a 37°C bath until initiating the labeling reaction. (3) The labeling reaction is carried out at room temperature by addition of the following reagents in the stated order: (i) Hz0 (to a total volume of 50 ~1); (ii) 10 ~1 oligo-labeling buffer (OLB buffer; see below): (iii) 2 ~1 of (10 mg/ ml) bovine serum albumin; (iv) DNA in agarose (up to 32.5 ~1); (v) 5 ~1 of [32P]dCTP (Amersham No. PB 10205, 3000-4000 Ci/ mmol, 10 j.&i/hl); (vi) 2 units of large fragment of Esckrichia co/i DNA polymerase I (BRL No. 80 12). Polymerization reaches a plateau in 2.5 h (30-50 ng DNA template), 5 h (lo30 ng), or overnight ( 10 ng or less). We generally use 20-30 ng DNA per reaction, but smaller amounts of DNA can be used if necessary. The reaction is stopped by addition of 200 ~1 of a solution containing 20 mM NaCl, 20 mM Tris-HCl (pH 7.5) 2 mM’ EDTA, 0.25% sodium dodecyl sulfate, 1 ,uM dCTP. Purification of the precursor nucleotide triphosphate from the labeled product is usually not necessary prior to hybridization. (4) OLB is made from the following components. Sdufion 0: 1.25 M Tris-HCl, 0.125 M MgCl?. at pH 8.0. Store at 4°C. S’olzltiotz ,3: 1 ml solution 0 + 18 ~1 2mercaptoethanol + 5 ~1 dATP, 5 ~1 dTTP. 5 ~1 dGTP (each triphosphate previously dissolved in TE (3 mM Tris-HCl, 0.2 mM EDTA,
267
ADDENDUM
pH 7.0) at a, concentration of 0.1 M). Store at -20°C. Solution B: 2 M Hepes, titrated to pH 6.6 with 4 M NaOH. Store at 4°C. SolutioFl C: Hexadeoxyribonucleotides (P-L No. 2 166) evenly suspended (this does not completely dissolve)in TE at 90 OD units/ ml. Store at -20°C. Mix solution A:B:C in a ratio of 100:250:150 to make OLB. Store at -20°C. Repeated freeze-thawing of OLB over a period of at least 3 months does not adversely affect it.
The bulk of the precursor [32P]dCTP is incorporated into acid-precipitable product under the above conditions. Single copy genes in higher eucaryotes can easily be detected on Southern blots in a 24-h exposure using probes constructed in this manner. ACKNOWLEDGMENTS This investigation was supported 35494, CA 31053, and GM 31676, tional Institutes of Health, DHHS.
by PHS Grants CA awarded by the Na-
ICAL
BIOCHEMISTRY
137, 266-267
(I 984)
ADDENDUM “A Technique
for Radiolabeling DNA Restriction Endonuclease Fragments to High Specific Activity” Volume
132. Number
1 (1983),
Volume 132, Number 1 (1983) in the article “A Technique for Radiolabeling DNA Restriction Endonuclease Fragments to High Specific Activity.” by Andrew P. Feinberg and Bert Vogelstein, pages 6- 13, we described a method for recovering DNA restriction endonuclease fragments from agarose gels and labeling them to high specific activity. We have since found that a modification of the labeling technique can be used to label DNA restriction fragments without purifying the DNA from the agarose. The modified technique described below eliminates the loss of DNA and time involved in recovering DNA fragments from agarose gels and can be used even with very small amounts of template DNA. (1) Plasmid or phage DNA is cleaved with an appropriate restriction endonuclease and the fragments are electrophoretically separated in one lane of a l-2% low gelling temperature agarose gel (Bio-Rad No. 162-00 17). The electrophoresis buffer is 40 mM Tris-acetate, 5 mM sodium acetate, 1 mM EDTA at pH 8.0. Generally, 4-8 hg of DNA can be run in a 24-mm’ lane without loss of resolution. After electrophoresis, the gel (10 X 14 X 0.3 cm) is stained for 20-30 min in a liter of HZ0 containing 0.2 pg/ml of ethidium bromide. (2) The desired band is excised cleanly and put into a preweighed 1.5-ml microcentrifuge tube. HZ0 is added at a ratio of 3 ml H20/g of gel. The tube is then placed in a boiling water bath for 7 min to dissolve the gel and denature the DNA. It is stored at -20°C. Pre-
0003-2697/84
$3.00
266
Pages 6-13
paratory to subsequent labelings, the gel is reboiled for 3 min and briefly stored (lo-60 min) in a 37°C bath until initiating the labeling reaction. (3) The labeling reaction is carried out at room temperature by addition of the following reagents in the stated order: (i) Hz0 (to a total volume of 50 ~1); (ii) 10 ~1 oligo-labeling buffer (OLB buffer; see below): (iii) 2 ~1 of (10 mg/ ml) bovine serum albumin; (iv) DNA in agarose (up to 32.5 ~1); (v) 5 ~1 of [32P]dCTP (Amersham No. PB 10205, 3000-4000 Ci/ mmol, 10 j.&i/hl); (vi) 2 units of large fragment of Esckrichia co/i DNA polymerase I (BRL No. 80 12). Polymerization reaches a plateau in 2.5 h (30-50 ng DNA template), 5 h (lo30 ng), or overnight ( 10 ng or less). We generally use 20-30 ng DNA per reaction, but smaller amounts of DNA can be used if necessary. The reaction is stopped by addition of 200 ~1 of a solution containing 20 mM NaCl, 20 mM Tris-HCl (pH 7.5) 2 mM’ EDTA, 0.25% sodium dodecyl sulfate, 1 ,uM dCTP. Purification of the precursor nucleotide triphosphate from the labeled product is usually not necessary prior to hybridization. (4) OLB is made from the following components. Sdufion 0: 1.25 M Tris-HCl, 0.125 M MgCl?. at pH 8.0. Store at 4°C. S’olzltiotz ,3: 1 ml solution 0 + 18 ~1 2mercaptoethanol + 5 ~1 dATP, 5 ~1 dTTP. 5 ~1 dGTP (each triphosphate previously dissolved in TE (3 mM Tris-HCl, 0.2 mM EDTA,
267
ADDENDUM
pH 7.0) at a, concentration of 0.1 M). Store at -20°C. Solution B: 2 M Hepes, titrated to pH 6.6 with 4 M NaOH. Store at 4°C. SolutioFl C: Hexadeoxyribonucleotides (P-L No. 2 166) evenly suspended (this does not completely dissolve)in TE at 90 OD units/ ml. Store at -20°C. Mix solution A:B:C in a ratio of 100:250:150 to make OLB. Store at -20°C. Repeated freeze-thawing of OLB over a period of at least 3 months does not adversely affect it.
The bulk of the precursor [32P]dCTP is incorporated into acid-precipitable product under the above conditions. Single copy genes in higher eucaryotes can easily be detected on Southern blots in a 24-h exposure using probes constructed in this manner. ACKNOWLEDGMENTS This investigation was supported 35494, CA 31053, and GM 31676, tional Institutes of Health, DHHS.
by PHS Grants CA awarded by the Na-