Primer information

Frequently using primer pairs for multi-loci analysis

Primer set(forward)/(reverse), annealing temperature

PCR condition

For TaKaRa Ex Taq (good at single copy genes and ribosomal genes, and mitochondrial gene)

98℃ 10 sec |

annealing ℃ 30 sec | 35-38 cycle

72℃ 1 min |

For TOYOBO KOD FX Neo (good at ribosomal genes, and mitochondrial gene)

94℃ 2 min

98℃ 10 sec |

annealing ℃ 30 sec | 35-38 cycle

68℃ 1 min |

SSU (nuclear small subunit (18S) ribosomal DNA)

NS1/NS4 42 °C (always using 46 to 48 °C)

NS1: GTAGTCATATGCTTGTCTC

NS4: CTTCCGTCAATTCCTTTAAG

ITS (nuclear ribosomal internal transcribed spacer region)

V9G/LR5, 53 °C

ITS5/ITS4 61.5 °C

V9G: TTACGTCCCTGCCCTTTGTA

LR5: TCCTGAGGGAAACTTCG

ITS5: GGAAGTAAAAGTCGTAACAAGG

ITS4: TCCTCCGCTTATTGATATGC

(Although previously ITS1 was used for standard primer for ITS, I feel something weakly amplified and sometimes amplified non-specific band, thus I dont recommend.)

LSU (nuclear large subunit (28S) ribosomal DNA)

LR0R/LR7 46 °C (always using 48 °C)

LR0R: ACCCGCTGAACTTAAGC

LR7: TACTACCACCAAGATCT

(Although sometimes LR5 is used for reverse primer, the product is too short for analysis, thus I dont recommend.

On the other hand, LR5 is good "sequencing primer", or ITS primer)

RPB1 (DNA-directed RNA polymerase II subunit, rpb1)

RPB1-Af/RPB1-Cr 53 °C

RPB1-Af: GARTGYCCDGGDCAYTTYGG

RPB1-Cr: CCNGCDATNTCRTTRTCCATRTA

RPB2(DNA-directed RNA polymerase II subunit, rpb2)

fRPB2-5F/fRPB2-7cR 58 °C

fRPB2-5F: GAYGAYMGWGATCAYTTYGG

fRPB2-7cR: CCCATRGCTTGYTTRCCCAT

Although some reviewer asked me too high for annealing temperature, but it is good for this region in my experiment (I dont know why).
Strongly recommend DO NOT USE KOD FX NEO, and USE TAQ POLYMERACE due to GC- and AT-rich of each half.

TEF1alpha (Translation elongation factor EF-1 alpha, don't recommend)

PCR pair Tm Cycle

EF1-728F/EF1-986R 58 °C 40 cycle

EF1-728F/TEF1LLErev 55 °C 40 cycle

EF1-983F/EF1-2218R 60 °C 40 cycle

Primer sequence type Reference

EF1-728F CATCGAGAAGTTCGAGAAGG PCR Carbone and Kohn (1999)

EF1-983F GCYCCYGGHCAYCGTGAYTTYAT PCR Rehner and Buckley (2005)

EF1-986R TACTTGAAGGAACCCTTACC PCR Carbone and Kohn (1999)

TEF1LLErev AACTTGCAGGCAATGTGG PCR Jaklitsch et al. (2006)

1567R ACHGTRCCRATACCACCSATCTT Seq Rehner and Buckley (2005)

EF1-2218R ATGACACCRACRGCRACRGTYTG PCR Rehner and Buckley (2005)

Reference

Carbone, I., & Kohn, L. M. (1999). A Method for Designing Primer Sets for Speciation Studies in Filamentous Ascomycetes. Mycologia, 91, 553–556. https://doi.org/10.2307/3761358

Jaklitsch, W. M., Komon, M., Kubicek, C. P., & Druzhinina, I. S. (2006). Hypocrea voglmayriisp. nov. from the Austrian Alps represents a new phylogenetic clade inHypocrea/Trichoderma. Mycologia, 97, 1365-1378. https://doi.org/10.1080/15572536.2006.11832743

Rehner, S. A., & Buckley, E. (2005). A Beauveria phylogeny inferred from nuclear ITS and EF1-alpha sequences: evidence for cryptic diversification and links to Cordyceps teleomorphs. Mycologia, 97, 84–98. https://doi.org/10.3852/mycologia.97.1.84

Google Sites

Report abuse