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Table 1 List of reagents and PCR steps to amplify chloroplast regions trnL-F, rps16, ndhF and nuclear regions ETS and ITS2

From: Optimization of DNA extraction and PCR protocols for phylogenetic analysis in Schinopsis spp. and related Anacardiaceae

PCR reagents

Final concentration

Chloroplast regions

Nuclear regions

H2O s&d

Buffer 5× (Promega)

DMSO 1 M (Sigma-Aldrich)

10 %

BSA 400 ng/μl (Promega)

1/1000

1/1000

Cl2Mg (Promega)

5 mM

5 mM

Primer F

1 μM

1 μM

Primer R

1 μM

1 μM

dNTP’s 1 mM (Promega)

200 μM

200 μM

DNA dil 1/10

25 ng

25 ng

Taq pol 5u/μl (Promega)

1.5–2 U

1.5–2 U

PCR steps

trnL-F/rps16

ndhF

ETS

ITS2

1—Initialization

2 min—97 °C

3 min—94 °C

10 min—95 °C

50 s—97 °C

2—Denaturation

1 min—94 °C

30 s—94 °C

1 min—94 °C

50 s—97 °C

3—Annealing

2 min—48/55 °C

1 min—48 °C

2 min—56 °C

50 s—53 °C

4—Extension

2 min—72 °C

50 s—72 °C

2 min—72 °C

1.5 min—72 °C

5—Elongation

16 min—72 °C

6 min—72 °C

16 min—72 °C

7 min—72 °C

 

[steps 2–3–4] × 30

[steps 2–3–4] × 35

[steps 2–3–4] × 35

[steps 2–3–4] × 35