Table 3 Master mix constituents and PCR conditions
From: An economical Nanopore sequencing assay for human papillomavirus (HPV) genotyping
PGMY PCR | ||
Master mix constituents (for single reaction) | ||
Reagent | Volume/μL | |
10X PCR buffer II (Applied Biosystems) | 5 | |
25 mM MgCl2 (Applied Biosystems) | 3 | |
PGMY primer mix (10 μM) | 1 | |
Human β-globin primer mix (5 μM) | 1 | |
10 mM dNTPs (Roche) | 1 | |
5 M betaine (Sigma) | 10 | |
AmpliTaq Gold DNA Polymerase (Applied Biosystems) | 0.25 | |
Molecular grade water (Sigma) | 23.75 | |
DNA | 5 | |
PCR conditions | ||
Temperature/oC | Time | No. of cycles |
95 | 9 min | 1 |
95 | 1 min | 40 (50% ramp) |
55 | 1 min | |
72 | 1 min | |
72 | 5 min | 1 |
15 | Hold | / |
MGP PCR | ||
Master mix constituents (for single reaction) | ||
Reagent | Volume/μL | |
10X PCR buffer II (Applied Biosystems) | 2.5 | |
25 mM MgCl2 (Applied Biosystems) | 1.5 | |
MGP primer mix (10 μM) | 0.5 | |
10 mM dNTPs (Roche) | 0.5 | |
AmpliTaq Gold DNA Polymerase (Applied Biosystems) | 0.1 | |
Molecular grade water (Sigma) | 14.9 | |
DNA | 5 | |
PCR conditions | ||
Temperature/oC | Time | No. of cycles |
95 | 10 min | 1 |
95 | 30 s | 5 |
42 | 30 s | |
72 | 30 s | |
95 | 30 s | 45 |
64 | 30 s | |
72 | 30 s | |
72 | 5 min | 1 |
15 | Hold | / |