A new metabarcoding approach to survey diversity at the species level of Arcellinida (Amoebozoa: Tubulinea)

First PCR

Primers:

Primer forward: LCO 1490 (5´ GGTCAACAAATCATAAAGATATTGG 3´)

Primer reverse: HCO 2198 (5´ TAAACTTCAGGGTGACCAAAAAATCA 3´)

Reagents for the PCR :

A	B
Polymerase	6 μL
BSA	1 μL
Primer forward	1 μL (10 μmol)
Primer reverse	1 μL (10 μmol)
Destilated water	1 μL
Sample (eDNA)	1 μL

Total reaction mix per sample= 10 μL

Thermocycler program:

A	B	C	D
	Temperature	Time	cycles
Initial denaturation	96 °C	5 min	1
Denaturation	94 °C	15 s	40
Annealing	40 °C	15 s
Extension	72 °C	90 s
Final extension	72 °C	10 min	1

Denaturation temperatures and times may vary depending on the polymerase used. This protocol was tested with the MyTaq™ Red Mix™ polymerase.

Gel electrophoresis:

Analyze the results of your PCR reaction via gel electrophoresis. The resultant fragment was 692 bp long:

Second PCR

Primers:

Primer forward: LCO 1490 (5´ GGTCAACAAATCATAAAGATATTGG 3´)

Primer reverse: ArCOIR (5´ CCACYNGAATGWGCTARAATACC 3´)

Reagents for the PCR :

A	B
Polymerase	12 μL
Primer forward	1 μL (10 μmol)
Primer reverse	1 μL (10 μmol)
Destilated water	6 μL
Sample (first PCR product)	1 μL

Total reaction mix per sample= 20 μL

Thermocycler program:

A	B	C	D
	Temperature	Time	cycles
Initial denaturation	96 °C	5 min	1
Denaturation	94 °C	15 s	40
Annealing	55 °C	15 s
Extension	72 °C	90 s
Final extension	72 °C	10 min	1

Denaturation temperatures and times may vary depending on the polymerase used. This protocol was tested with the MyTaq™ Red Mix™ polymerase.

Gel electrophoresis:

Analyze the results of your PCR reaction via gel electrophoresis. The resultant fragment was 407 bp long