If you need to cite these protocols, please use: Lyne et al. (2003). BMC Genomics 4:27 (PDF).

Gene-specific primers

  • Primer length: 18-22 bp (+8 bp universal sequence for the forward primers)
  • Annealing temperature: 58-62ºC
  • GC content: 40-60%
  • Product length: 200-500 bp
  • Reverse primers <2500 bp from 3' ends of genes

The parameters above were used for most primers. In a few cases primers deviating from some of these parameters had to be chosen.
Primers were selected using the Primer3 program together with customized scripts written in Perl. Whenever possible, primers were chosen such that the resulting product had a BLAST score <400 with other sequences within the S. pombe genome.
Primers were ordered from Genset.

PCR (1st round)

Ingredients Volume/20 µL reaction
Template DNA 0.5 µL
10x Buffer 2 µL
dNTP mix (10mM each) 0.25 µL
Amplitaq DNA Polymerase 0.125 µL
Primers (10µM each) 1 µL
ddH2O 16.125 µL

Genomic DNA (prepared using a simple glass bead protocol) is used as template. For genes with all exons <250 bp, pooled cDNA libraries are used as template.
10 x Buffer: 500mM KCl, 50mM Tris 8.5, 25mM MgCl2.

PCR reactions are performed in 96 well plates in a Tetrad thermocycler under the following conditions:

  • 95ºC 3 min
  • 35 cycles of 94ºC 30 sec, 60ºC (-0.3ºC/cycle) 45 sec, 72ºC 45 sec
  • 72ºC 5 min

PCR (2nd round)

Ingredients Volume/100 µL reaction
Template DNA 1 µL
Universal primers (100 µg/ml) 4 µL
Gene specific reverse primer (10µM) 4 µL
10x Buffer 12 µL
dNTP mix (10mM each) 3 µL
Amplitaq DNA polymerase 0.75 µL
ddH2O 75.25 µL

The PCR reaction mix of the first round is directly used as the template for the 2nd round of PCR.

PCR reactions are performed under the following conditions:

  • 94ºC 1 min
  • 38 cycles of 94ºC 30 sec, 55ºC 45 sec, 72ºC 45 sec
  • 72ºC 5 min

Quality Control and Failures

All PCR reactions are checked on 2.5% agarose 1x TBE slab gels for single strong bands of expected size. Typically, the failure rate is less than 3%. Failures are repeated and new primer sequences are ordered in the cases where PCR reactions failed repeatedly.