4. Run the PCR in a thermocycler by using the conditions suggested by the polymerase manufacturer. We typically denature
the template DNA at 98
C for 30 s, perform 25 cycles of
amplification (denaturation at 98
C for 30 s, annealing at the
calculated Tm for 15 s and elongation at 72
C for 60 s in the
case of a 1 kb gene) followed by a final incubation at 72
C for
10 min.
5. Load 1/10 of the PCR volume on a 1% agarose gel in parallel
with a molecular weight marker to verify that the PCR amplification resulted in a single band of the expected size.
Fig. 6 Modification of an existing construct. In this example, RF cloning is used to introduce a cleavable affinity
tag (3C-twinstrep) at the 3
0
extremity of the MAT1 cDNA which is part of a multigene expression construct. The
sequence of the modified plasmid where the existing part (in blue) and the insert (in green) are shown. The
hybrid primers MAT1-TwinStrep-FW and MAT1-TwinStrep-RV used to amplify the insert (3C cleavage site and
Twin-Strep tag) and introduce 20–22 bp overhangs matching the MAT1 gene at the insertion point (immediately before the Stop codon) are also displayed
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Paola Rossolillo et al.