COI
PCR reaction condition (final volume 20 μL)
PCR program
Initial denaturation
Total 3 step cycling
Denaturation
Annealing
Extention
Final elongation
Buffer
(5Prime, Gaithersburg, USA),
16SrRNA
94°C for 3 minutes
40 cycles
94°C for 30 seconds
51°C for 30 seconds
72°C for 35 seconds
72°C for 5 minutes
45 cycles
94°C for 25 seconds
57.5°C for 15 seconds
72°C for 2 seconds
72°C for 10 minutes
2μL of 10× buffer
MgCl2
1.5 mM
dNTPs
(dNTPmix, EurocloneS.p.A - Life Sciences
Division, Pavia, Italy)
200 μM each
BSA (Purified BSA 100×, New England
BIOLABS® Inc. Ipswich, MA, USA),
25 ng/μL
Primers
300 nM
PerfectTaq DNA Polymerase (5Prime,
Gaithersburg, USA),
1.25 U
DNA template
100 ng
DNase free water (Water Mol. Bio. Grade,
DNase–RNase and Protease free, 5Prime
GmbH, Hamburg, Germany).
Up to final volume
Table 4SM. Standard PCR program and reaction condition used for the amplification of the COI and 16SrRNA gene. The amplifications were carried on a LifePro™ Gradient
Thermal Cycler (BIOER TECHONOLOGY CO., LTD).