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. 2010 Feb 16;10:45. doi: 10.1186/1471-2148-10-45

Table 2.

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

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

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.