PCR reagents | Final concentration | |
---|---|---|
Chloroplast regions | Nuclear regions | |
H2O s&d | ||
Buffer 5× (Promega) | 1× | 1× |
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 |