The program gives five possible pairs of primers of user specified length, upstream and downstream of the target gene. The program produces primers as described in Bähler et al. Yeast (1998) 14: 943


Name of Gene - Name of the gene you wish to delete. Case insensitive. Valid gene names are those found in S.pombe GeneDB.
Length of Primer target sequence - Required length of primer pairs, excluding plasmid specific sequence.
Plasmid used as a PCR template - A plasmid specific sequence is added to the primers.
Primer increment - The first set of primers given in the output (PRIMER_FORWARD_1 and PRIMER_REVERSE_1) will be directly adjacent to the ORF. Four subsequent sets of primers are also shown, and these sets are further away from the ORF at a user specified distance. For example, if the primer increment is 40, the second set of primers will begin 40bp before and after the ORF, the 3rd set will be placed 80bp before and after the ORF, the 4th set 120bp away and the 5th set 160bp away. This allows for a choice of primers with varying positions and base composition. 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_1 and PRIMER_REVERSE_1 give primers of the specified length directly adjacent to the ORF. PRIMER_FORWARD_2 and PRIMER_REVERSE_2 give primers of the same length a specified distance from the ORF as explained under 'primer increment' in the input above.
The plasmid specific sequence follows the primer sequence. If no 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 that the forward primer ends upstream of the ORF, or that the reverse primer ends downstream of the ORF.