2 Enzymatic Modification of DNA and RNA
39
• T4 polynucleotide kinase (usually 10 U/Jll)
• Ultrapure water
• EDTA 0.5 M pH8.0
Procedure
1. On ice, pipet the following into a 1.7 ml or 0.5 ml reaction tube:
Prim er
20 )ll
50 pmol
lOx buffer
5 )ll
y- 32 P ATP
15 )ll
150 )lCi, 50 pmol
T4 kinase
2 )ll
20 u
Water
8 )ll
Note: The reaction can be scaled down ifless primer is needed. Labeling of 1
pmol primer can be performed in as little as 5 Jll reaction volume. The primer should then be added as 1 pmol/2 Jll solution. Use only 1J.1ll0x buffer,
1.5 111 y- 32 P ATP, 10 U T4 polynucleotide kinase.
2. Incubate at 37°C for 30 min.
Note: If small volumes are used, the reaction may directly be used for subsequent sequencing reactions without further purification. In this case, skip
the following steps.
3. Add 2 111 EDTA solution to stop the reaction.
4. Purify the DNA by using a chaotropic buffer/silica-gel spin column system (e. g. Qiagen).
5. Elute the DNA in 30 111 water.
Note: Unincorporated nucleotides can also be eliminated by small scale gel
filtration (e. g. QuickSpin™, Boehringer).
T4 kinase