The forward primer added LVPRGS thrombin cleavage site to the N-terminal of the CBMCBHI sequence.
3. Next, layer face powder on top of your eye primer, adding neutral contour to the outer corner of your eye to give it a little definition.
The 3' primer added two stop codons and a BamHI site.
Reactions with only one primer added served as negative controls.
The addition of the M13 tail sequence to the 5′ end of each forward SSR primer allows the amplicons to be visualized by the fluorescence emitted by the "fluorophore" bound to the M13 primer (third primer added to the PCR).
Often, these reactions were performed "semi-nested" with a third primer added at 0.1X the concentration of each of the other two (PGLU-TAT for Cytb, and cytochrome oxidase I primer MVZ 202 for ND2; Table 1).
For entry clone generation to be transferred to N-terminal tag expression vectors the 5'-linkers for the forward and reverse primers were 5' AAAAAGCAGGCTCT 3' and 5' AGAAAGCTGGGTTCTA 3' respectively with the reverse primer adding a stop codon.
The supernatant was then discarded and 0.3 μΜ of the PSQ primer added annealed to the captured strand and incubated at 80°C for 2 minutes on the PSQ plate.
These EST-SSR primers add value to a recently expanding set of EST-SSRs being made available for Eucalyptus, increasing the variety of genes that can be investigated in novel population and conservation genetic studies of nonmodel species.
The primers add an ATG codon to the start of the open reading frame and the HC-Pro gene was cloned as a KpnI- XhoI fragment into pMT/V5-HisA (Invitrogen) to produce plasmid pMT-HC-Pro.
Selective amplification primers added two or three additional bases to the pre-selective sequences to further reduce the number of amplified fragments (Table 1).
