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Table 2 Standard PCR and sequencing primers, primer combinations and annealing temperatures used

From: Evolution of plant RNA polymerase IV/V genes: evidence of subneofunctionalization of duplicated NRPD2/NRPE2-like paralogs in Viola (Violaceae)

Region

Forward primer

Reverse primer

Annealing temperature

ex5-in6 (PCR 1)

5F2898: TTGACAGCCTYGATGATGAT

Svex7R3420: ATCTTGAAAATCCAGCCC

52°C

ex5-in6

5F3062: AATGATGASGGGAAGAATTTTGC

Svex7R3420

52°C

ex6-ex7 (PCR 2)

vex6F3263: GYCARCTYCTTGAGGCTGC

7R3883: ATVCCCATGCTGAAKAGCTCYTG

59°C

ex5-ex6

5F2898

vex6R3371: YMTCRACACTGGGAGTGGAG

54°C

ex5-ex6

5F3062

vex6R3371

57°C

ex6-in6

vex6F3263

Svex7R3420

52°C

ex7

vex7F3418: GGCTGGATTTTCAAGATGG

7R3883

55°C

  1. PCR mix: 20 to 40 μl reactions; 0.2 mM dNTPs, 0.25 μM of each of the primers, 1× Phusion HF buffer, 0.008 U/μl Phusion polymerase. The PCR conditions were as follows: initial denaturation at 95°C for 30 s followed by 35 cycles of 95°C for 9 s, annealing at a temperature specified below for 30 s, and 72°C for 30 s. The PCR ended with 7:30 minutes at 72°C and subsequent soak at 10°C.