output gives one upstream/forward primer and five possible downstream/reverse primers for a specified S.pombe gene for C-terminal tagging.

The program produces primers as described in Bähler et al. Yeast (1998), 14: 943


Name of Gene - Name of the gene you wish to amplify using PCR. Case insensitive. Valid gene names those found in S.pombe GeneDB.
Length of target sequence - Required length of primer sequences, excluding the plasmid specific sequence. The forward primer will always be inside the ORF, ending just before the STOP codon.
Plasmid used as a PCR template - A plasmid specific sequence added to the primers.
Primer increment - The first reverse primer given in the output PRIMER_REVERSE_1 will be directly adjacent to the ORF. Four subsequent primers are given also, and they are positioned away from the ORF at user specified increments. For example, if the primer increment is 40, the second primer will begin 40bp after the ORF, the 3rd will be placed 80bp after the ORF, the 4th primer 120bp away and the 5th 160bp away. This allows for a choice of primers should the initial primer not be suitable. The primers may overlap.


Chromosome - 1,2 or 3
Gene Length - length of ORF including introns.
Primers The primer output is presented in the form [PRIMER_NAME] = [PRIMER SEQUENCE]- [PLASMID SPECIFIC SEQUENCE] [GC CONTENT] [TM] [DISTANCE FROM ORF] for each primer.
PRIMER_FORWARD - User specified length, inside the ORF, ending just before the STOP codon.
PRIMER_REVERSE_NUMBER - see primer increment above
The plasmid specific sequence follows the primer sequence. If no specific plasmid is selected, "- 0" is printed to indicate this.
GC Content - GC content as a percentage of the total nucleotide content of the primer plus any plasmid specific sequence.
TM - Tm for the primer as given by the equation

Tm = 81.5 + 16.6(log10([Na+])) + 0.41*(%GC) - 600/length

(Sambrook, Fritsch and Maniatis, Molecular Cloning, p11.46 1989, CSHL Press)
[Na+] is assumed to be 0.2M.

For small oligos (~20bp) this may be inaccurate. A more accurate Tm can be obtained using thermodynamic methods as described in Breslauer, Frank, Bloeker and Marky. Proc. Natl. Acad. Sci USA,8; 3746.
Distance from ORF - number of base pairs primer that the reverse primer ends downstream of the ORF.