Chimera construction or insertion with a short DNA fragment. To replace a short stretch of DNA, such as a DNA fragment encoding a transmembrane region of a nAChR subunit for chimera construction, or to insert a short tag, such as FLAG-tag, into some part of a protein, amplification of the insert is not necessary. In this case, the insert can be directly included in two primers for single PCR amplification of the cDNA along with the vector. The forward primer starts immediately downstream of the insertion site and has a tail with the 3' part of the insert. The reverse primer starts immediately upstream of the insertion site and has a tail with 5' part of the insert. Two primers only require ~16-base overlap. Thus, for a 120 bp insertion, each primer needs to have a 68-base tail for insertion and ~17-22 bases for annealing (depending on the GC content). The total length of each primer will be about 85-90 bases. DpnI digestion and transformation are the same as in Figure 1.
Li et al. BMC Biotechnology 2011 11:92 doi:10.1186/1472-6750-11-92