John M. S. Bartlett.pdf - Bio-Nica.info

T-Linker Strategy 477
Fig. 2. Using a T-linker to add a “histidine tag” to one end of a PCR product. (A) Oligonucleotide
design. The NdeI site allows the product to be cloned into an appropriate expression
vector, in which translation begins at the ATG codon included in this site. The amino acids that
make up the histidine tag are shown, and the reading frame is indicated by vertical bars between
codons. The NotI site in HisTL-A is in parentheses because it would not be expected to cut so
close to the end of a DNA molecule (although it should be possible to use it if the primer were
phosphorylated and the products ligated to form concatamers). Its main purpose is to serve as
a clamp to allow efficient cutting at the NdeI site. (B) Sequence at the junction between the
HisT-linker and the 5′ end of the PCR-amplified sequence. The 5′ primer begins at the first base
in a codon, to put the sequence in frame with the histidine tag. (C) Sequence at the 3′ junction.
The 3′ primer begins with ta; the complementary ta in the other strand is converted to a stop
codon “taa” when the extra a is added by Taq polymerase. Because the 5′ primer begins with c,
the HindIII site is completed only at the 3′ end of the molecule. This allows directional cloning
of the product, and removal of the T-linker sequences from the 3′ end.