Modification of Nucleic Acids and Oligonucleotides

Remove the supernatant and wash the pellet with 70 and 100 percent ethanol, centrifuging after each wash. 

Re-dissolve the labeled DNA pellet in water and store at —20°C until used. 

Denature 1 pg of probe DNA (single stranded) with 5 pg of random hexanucleotide primers by boiling for 5 minutes and then rapidly chill on ice. Incubate at least 10 minutes to allow the primers to hybridize to random sites within the probe DNA. 

Add to a tube on ice, 5 pi of 10X random priming buffer (0.5M Tris, 0.1 M MgCl2, lOmM 2-mercaptoethanol, pH 6.6, containing 0.5mg/ml BSA), lpl each of 3 types of unmodified deoxynucleoside triphosphates (dNTPs at 100 pM concentration), lpl of a labeled dNTP (at 100 pM), plus 48 pi of water. Then add 2 pi of DNA polymerase (5-10 units). 

Combine the probe DNA/hexanucleotide preparation with the reaction solution and incubate for 2 hours at 37°C. 

Purify the labeled DNA from excess reactants by precipitation. Add 20 pi of 4 M LiCl and 500 pi of ethanol (chilled to —20°C). Mix well. 

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Modification of Nucleic Acids and Oligonucleotides with Cystamine... 

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