BsaI-HF®v2

Protocol tips

Upstream tips	Protocol tips	Downstream tips
	All Golden Gate reactions were performed in a total volume of 15 µl. The final reaction volume contained 1-fold concentrated T4 ligase buffer (Promega, Madison, US). Prepared reaction mixtures (ligase buffer, acceptor plasmid, insert(s)) was adjusted to 13.5 µl with ddH2O. In a final step, the corresponding enzymes were quickly added. First, a volume of 0.5 µl of the respective restriction enzyme BbsI (5 units; ThermoFisherScientific, Waltham, US) or BsaI-HF®v2 (10 units; New England Biolabs, Ipswich, US) and then 1 µl (1–3 units) of T4 ligase (Promega, Madison, US) was added. Golden Gate reactions were carried out by default under following conditions: a) Enzymatic restriction 37 °C (2 min) [40 passes]; b) Ligation 20 °C (5 min) [40 passes] and c) enzyme inactivation: 80 °C (20 min).

Protocol tips

All Golden Gate reactions were performed in a total volume of 15 µl. The final reaction volume contained 1-fold concentrated T4 ligase buffer (Promega, Madison, US). Prepared reaction mixtures (ligase buffer, acceptor plasmid, insert(s)) was adjusted to 13.5 µl with ddH2O. In a final step, the corresponding enzymes were quickly added. First, a volume of 0.5 µl of the respective restriction enzyme BbsI (5 units; ThermoFisherScientific, Waltham, US) or BsaI-HF®v2 (10 units; New England Biolabs, Ipswich, US) and then 1 µl (1–3 units) of T4 ligase (Promega, Madison, US) was added. Golden Gate reactions were carried out by default under following conditions: a) Enzymatic restriction 37 °C (2 min) [40 passes]; b) Ligation 20 °C (5 min) [40 passes] and c) enzyme inactivation: 80 °C (20 min).

Publication protocol

"All Golden Gate reactions were performed in a total volume of 15 µl.
Te fnal reaction volume contained 1-fold concentrated T4 ligase bufer (Promega, Madison, US). Prepared
reaction mixtures (ligase bufer, acceptor plasmid, insert(s)) was adjusted to 13.5 µl with ddH2O. In a fnal step,
the corresponding enzymes were quickly added. First, a volume of 0.5µl of the respective restriction enzyme BbsI
(5 units; TermoFisherScientifc, Waltham, US) or BsaI-HF®v2 (10 units; New England Biolabs, Ipswich, US) and
then 1µl (1–3 units) of T4 ligase (Promega, Madison, US) was added. Golden Gate reactions were carried out by
default under following conditions: a) Enzymatic restriction 37°C (2min) [40 passes]; b) Ligation 20 °C (5min)
[40 passes] and c) enzyme inactivation: 80 °C (20min)."

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Manufacturer protocol

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