For cloning DNA fragments by ligating with dephosphorylated vector DNA, the fragments should have phosphates on their 5' termini. Since phosphorylation of PCR products by T4 polynucleotide kinase is inefficient, primers should be phosphorylated prior to PCR reaction.
1. Phosphorylation of PCR primers.
Mix the following solutions for each primer required for PCR. The 10x T4 PKase buffer contains 0.7 M Tris-Cl (pH 7.6), 0.1 M MgCl2 and 50 mM dithiothreitol.
primer (100 pmol/ul)
10x T4 PKase buffer
10 mM ATP
T4 polynucleotide kinase
Incubate for 1 hour at 37 degrees. Store frozen at -20 degree.
Mix the following solutions.
10x PCR buffer
phosphorylated primer (forward)
phosphorylated primer (reverse)
dNTP mix (2.5mM each)
thermostable DNA polymerase
Perform polymerase chain reaction, then purify PCR product(s) by agarose gel electrophoresis or appropriate cartridge.
3. Polishing of termini of PCR product.
(This step is not necessary if the thermostable DNA polymerase used in the PCR in step 2 had proof-reading activity.)
To the PCR product dissolved in 20-50 ul of TE, add 1/8 vol each of 10x buffer M (100 mM Tris-Cl (pH7.5), 100 mM MgCl2, 500 mM NaCl, 10 mM DTT) and dNTP mix (2.5 mM each of 4dNTPs). Add 2 units of T4 DNA polymerase. Incubate for 30 min at room temperature.
4. Purify DNA by phenol-extraction followed by ethanol-precipitation or spun-column. The DNA is ready for ligation with blunt-ended dephosphorylated vector.