Component (concentration) (supplier) | Volume ( μL) per 10 μL reaction |
---|---|
Autoclaved PCR-grade MilliQ™water | 2.8 |
10 × ThermoPol reaction buffer (containing 2 mM MgCl2) (New England Biolabs) | 1 |
*25 mM MgCl2 (4 mM final) (TaKaRa) | 1.6 |
10 mM dNTP’s (0.8 mM final) (TaKaRa) | 0.8 |
FIP 20 μM (0.8 μM final) (Invitrogen) | 0.4 |
BIP 20 μM (0.8 μM final) (Invitrogen) | 0.4 |
F3 10 μM (0.2 μM final) (Invitrogen) | 0.2 |
B3 10 μM (0.2 μM final) (Invitrogen) | 0.2 |
5 M Betain (0.8 M final) (Sigma) | 1.6 |
Bst DNA polymerase 8 U/μL (0.32 U/μL final) (New England Biolabs) | 0.4 |
AMV reverse transcriptase 10 U/μL (0.05 U/μL final) (Promega) | 0.05 |
DNA/RNA template or Crude RNA extract | 0.6 |