II. PCR Amplifications by Locus

A. PCR of D3 LSUrDNA (AmpliTaq)

1. Reaction mix Reaction volume: 25µl 50µl Total Sample: File name:

Chemicals	Stock	Final25µl	Per reaction 25µl	Total volume	Per reaction 50µl	Total volume	12 reaction 50µl	Check
RNase/DNase free water			14.7µl		29.4µl		352.8µl
10X PCR reaction buffer	10X	1X	2.5µl		5µl		60µl
MgCl2	25mM	2.5mM	2.5µl		5µl		60µl
Forward primer D3a	10µM	0.4µM	1µl		2µl		24µl
Reverse primer D3b	10µM	0.4µM	1µl		2µl		24µl
dNTPs	10mM	0.2mM	4x0.5µl		4x1µl		4x12µl
AmpliTaq (Applied Biosystems)	5 unit/µl	1.5 unit	0.3µl		0.6µl		7.2µl
DNA			1µl		2µl		2µl
Total			25µl		50µl		50µl

Note: D3a primer (5’ GACCCGTCTTGAAACACGGA 3’), D3b primer (5’ TGCGAAGGAACCAGCTACTA 3’)

1. Thaw all the reagents completely, burst spin and centrifuge before using. Work on the ice.
2. Dispense 24µl mix except for DNA to each PCR tube (0.5ml).
3. Add 1µl of DNA sample to the tubes.
4. Place in BioRad I Cycler thermal cycler.
5. Pour 25ml 1.5% agarose gel. Run it at 96V for 20 min or_____V for ______min.

2. PCR program (D3 55 PCR): 3 hours 10 mins in total. Start: _____finish: ______.

1. Step 1: 95ºC for 5 min Cycle number: 1
2. Step 2: 95ºC for 25 sec

55ºC for 30 sec Cycle number: 35

72ºC for 2.5 min

3. Step 3: 72ºC for 10 min Cycle number: 1
4. Step 4: Hold temperature: 4ºC

B. D2/D3 rDNA PCR (AmpliTaq)

1. Reaction mix Reaction volume: 25µl 50µl Total Sample: File name:

Chemicals	Stock	Final25µl	Per reaction 25µl	Total volume	Per reaction 50µl	Total volume	12 reaction 50µl	Check
RNase/DNase free water			13.7µl		27.4µl		328.8µl
10X PCR reaction buffer	10X	1X	2.5µl		5µl		60µl
MgCl2	25mM	2.5mM	2.5µl		5µl		60µl
Forward primer D2a	10µM	0.4µM	1µl		2µl		24µl
Reverse primer D3b	10µM	0.4µM	1µl		2µl		24µl
dNTPs	10mM	0.2mM	4x0.5µl		4x1µl		4x12µl
AmpliTaq	5 unit/µl	1.5 unit	0.3µl		0.6µl		7.2µl
DNA			1µl		2µl		2µl
Total			25µl		50µl		50µl

Note: D2a primer (5’ ACAAGTACCGTGAGGGAAAGT 3’), D3b primer (5’ TGCGAAGGAACCAGCTACTA 3’)

Ferg-ID2B primer (5’ AGTAACCTCTTGCACCAAAC 3’), D2B primer (5’ GACCCGTCTTGAAACACGGA 3’)

1. Thaw all the reagents completely, burst spin and centrifuge before using. Work on the ice.
2. Dispense 24µl mix except for DNA to each PCR tube (0.5ml).
3. Add 1µl of DNA sample to the tubes.
4. Place in BioRad I Cycler thermal cycler.
5. Pour 25ml 1.5% agarose gel. Run it at 96V for 20 min

2. PCR program (D3 55 PCR): 3 hours 10 mins in total. Start: ______finish: _______.

1. Step 1: 95ºC for 5 min Cycle number: 1
2. Step 2: 95ºC for 30 sec

55ºC for 40 sec Cycle number: 35

72ºC for 2 min

3. Step 3: 72ºC for 10 min Cycle number: 1
4. Step 4:Hold temperature: 4ºC

C. 18S rDNA PCR (Dynazyme)

1. Reaction mix Reaction volume: 25µl 50µl Total Sample: File name:

Chemicals	Stock	Final25µl	Per reaction 25µl	Total volume	Per reaction 50µl	Total volume	16 reaction 25µl	Check
RNase/DNase free water			15.5µl		31µl		248µl
10X PCR reaction buffer	10X	1X	2.5µl		5µl		40µl
MgCl2	50mM	2.5mM	0.75µl		1.5µl		12µl
Forward primer	10µM	0.63µM	1.25µl		2.5µl		20µl
Reverse primer	10µM	0.63µM	1.25µl		2.5µl		20µl
dNTPs	10mM	0.2mM	4x0.5µl		4x1µl		4x8µl
Taq	5 unit/µl	2.5 unit	0.5µl		1µl		8µl
DNA			1µl		2µl		1µl
Total			25µl		50µl		50µl

Note: 18S-G18S4 primer (5’ GCTTGTCTCAAAGATTAAGCC 3’), 18S-18P primer (5’ TGATCCWKCYGCAGGTTCAC 3’) or Bursaphelenchus specific: 18SF-Burs: ATGCATGTCTAAGTGGAGTATTATA 18SR-Burs: CTACGGCTACCTTGTTACGACTTTT

1. Thaw all the reagents completely, burst spin and centrifuge before using. Work on the ice.
2. Dispense 19µl mix except for DNA to each PCR tube (0.2ml).
3. Add 1µl of DNA sample to the tubes.
4. Place in BioRad I Cycler thermal cycler.
5. Pour 25ml 1.5% agarose gel. Run it at 96V for 20 min or_____V for ______min.

2. PCR program (47 PCR40): 3 hours 10 mins in total. Start: ______finish: _______.

1. Step 1: 95ºC for 4 min Cycle number: 1
2. Step 2: 95ºC for 30 sec

42ºC for 30 sec Cycle number: 40

72ºC for 3 min

c. Step 3: 72ºC for 10 min Cycle number: 1

d. Step 4:Hold temperature: 4ºC

D. COI- mtDNA PCR by AmpliTaq polymerase

1. Reaction mix Reaction volume: 25µl 50µl Total Sample: File name:

Chemicals	Stock	Final25µl	Per reaction 25µl	Total volume	Per reaction 50µl	Total volume	12 reaction 50µl	Check
RNase/DNase free water			14.7µl		29.4µl		352.8µl
10X PCR reaction buffer	10X	1X	2.5µl		5µl		60µl
MgCl2	25mM	2.5mM	2.5µl		5µl		60µl
Forward primer	10µM	0.4µM	1µl (10pmol)		2µl		24µl
Reverse primer	10µM	0.4µM	1µl(10pmol)		2µl		24µl
dNTPs	10mM	0.2mM	4x0.5µl		4x1µl		4x12µl
AmpliTaq (Applied Biosystems)	5 unit/µl	1.5 unit	0.3µl		0.6µl		7.2µl
DNA			1µl		2µl		2µl
Total			25µl		50µl		50µl

Note: COI-F1 primer (5’ CCTACTATGATTGGTGGTTTTGGTAATTG 3’), COI-R1 primer (5’ GTTGAGGGAAAAATGTTAAATTAACTCC 3’)

COI-F2 primer (5’ CCTGTCTTGGCTGGTGCTATTAC 3’), COI-R2 primer (5’ GTAGCAGCAGTAAAATAAGCACG 3’)

COI-F3 primer (5’ TGAGCTCACCATATGTATACAGTAGG 3’), COI-R3 primer (5’ CAATGTAGAGCAAAAATAACTAAATC 3’)

1. Thaw all the reagents completely, burst spin and centrifuge before using. Work on the ice.
2. Dispense 24µl mix except for DNA to each PCR tube (0.5ml).
3. Add 1µl of DNA sample to the tubes.
4. Place in BioRad I Cycler thermal cycler.

2. PCR program (51 CO): 3 hours 10 mins in total. Start: ______finish: _______.

1. Step 1: 95ºC for 5 min Cycle number: 1
2. Step 2: 95ºC for 30 sec

51ºC for 45 sec Cycle number: 35

72ºC for 2 min

3. Step 3: 72ºC for 10 min Cycle number: 1
4. Step 4: Hold temperature: 4ºC