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HEINZ-ULRICH G. WEIER
In the second amplification stage, 20 f..ll of the reaction product are
resuspended in a 200 f..ll Taq amplification reaction buffer and amplified
with primer JUNI5 (5-CCCAAGCTTGCATGCGAATTC-3) with the following PCR conditions: denaturation at 94°C for I min, primer annealing
at 50°C for I min, and extension at 72 °c for 2 min, repeated for 35 cycles.
After precipitation of the PCR products in 1.2 vol of isopropanol, the products are resuspended in 30 f..ll of TE buffer. Subsequently, 1.5 f..ll of this
solution is labeled in a 25 f..ll random priming reaction incorporating FITC12-dUTP or biotinylated dNTPs.
Subprotocol 6
Probe labeling via random priming and hybridization
Labeling of DNA by random priming is a reliable method and, in our laboratory, is applied routinely to label DNA fragments from 100 bp to several hundred kb. The procedure involves an initial thermal denaturation
of the DNA to allow the random oligonucleotides ('primers') to anneal.
Thus, restriction or hydrolysis oflarge molecules is not necessary. Several
companies now offer kits for random priming reactions. Slight differences
exist with regard to enzyme activity, amount of random primers and cost
per reaction.
Procedure
Measurement of DNA concentration
The concentration ofPCR products can be estimated from the agarose gels
run to confirm target amplification. If a sufficient amount of clonal or
genomic DNA is available, one or two microliters can be used to accurately
determine the concentration using Hoechst 33258 fluorometry using a
TKIOO fluorometer (Pharmacia).
Random priming
1. Add 250 ng of DNA to water to a final volume of7 f..ll in a O.5-ml microcentrifuge tube.
2. Boil DNA at 100°C for 5 min, then quickly chill on ice.
HEINZ-ULRICH G. WEIER
In the second amplification stage, 20 f..ll of the reaction product are
resuspended in a 200 f..ll Taq amplification reaction buffer and amplified
with primer JUNI5 (5-CCCAAGCTTGCATGCGAATTC-3) with the following PCR conditions: denaturation at 94°C for I min, primer annealing
at 50°C for I min, and extension at 72 °c for 2 min, repeated for 35 cycles.
After precipitation of the PCR products in 1.2 vol of isopropanol, the products are resuspended in 30 f..ll of TE buffer. Subsequently, 1.5 f..ll of this
solution is labeled in a 25 f..ll random priming reaction incorporating FITC12-dUTP or biotinylated dNTPs.
Subprotocol 6
Probe labeling via random priming and hybridization
Labeling of DNA by random priming is a reliable method and, in our laboratory, is applied routinely to label DNA fragments from 100 bp to several hundred kb. The procedure involves an initial thermal denaturation
of the DNA to allow the random oligonucleotides ('primers') to anneal.
Thus, restriction or hydrolysis oflarge molecules is not necessary. Several
companies now offer kits for random priming reactions. Slight differences
exist with regard to enzyme activity, amount of random primers and cost
per reaction.
Procedure
Measurement of DNA concentration
The concentration ofPCR products can be estimated from the agarose gels
run to confirm target amplification. If a sufficient amount of clonal or
genomic DNA is available, one or two microliters can be used to accurately
determine the concentration using Hoechst 33258 fluorometry using a
TKIOO fluorometer (Pharmacia).
Random priming
1. Add 250 ng of DNA to water to a final volume of7 f..ll in a O.5-ml microcentrifuge tube.
2. Boil DNA at 100°C for 5 min, then quickly chill on ice.