gives five possible pairs of primers of user specified length.
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 Primer Target Sequence - The required length for the primers, excluding plasmid specific sequence. Default 80bp.
Primer increment - The first forward of primer given in the output (PRIMER_FORWARD_1) will be directly adjacent to the ORF. Four subsequent forward primers are also given and they are further away from the ORF in user specified increments. For example, if the primer increment is 40, the second forward primer will begin 40bp before and after the ORF, the 3rd primer will be placed 80bp before and after the ORF, the 4th primer 120bp away and the 5th primer 160bp away. This allows for a choice of primers with varying positions and base composition. The primers may overlap. Negative increment values will give primers inside the ORF.
N-Terminal Tag-Sequence specific to a choice of N-terminal tags added. See Bähler et al 1998 for details.


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.
The reverse primer is internal to the ORF and ends just before the start codon. If an N-Terminal tag has been chosen, the next 3 bases are shown in brackets in the output. They should be [CAT], the reverse complement of the start codon ATG.
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 that the forward primer ends upstream of the ORF.