FastDigest Eco31I (IIs class)

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	Reactions were set up using 20 fmol of each Golden Gate entry vector and the destination vector, 10 U of BsaI Restriction Enzyme (Thermo, Fisher Fast Digest Enzyme Eco31I) and 30 U of T4 DNA Ligase (Thermo, Fisher) in 2 µl 10x ligation buffer (10x ligation buffer was prepared by supplementing the Thermo, Fisher FastDigest buffer with 10mM dATP and 100mM DTT) to a final reaction volume of 20 µl with ddH2O. It is crucial for the reactions that the entry vectors are purified and diluted in pure water since buffer components might inhibit the Golden gate assembly. All reactions were incubated using the following cycling conditions: 2 min at 37 °C, 5 min at 20°C for 50 cycles, then 5 min at 50°C and 5 min at 80°C. 10µl of the reaction mix were used to transform chemical competent cells (Top10 strain from Life Technologies).

Protocol tips

Reactions were set up using 20 fmol of each Golden Gate entry vector and the destination vector, 10 U of BsaI Restriction Enzyme (Thermo, Fisher Fast Digest Enzyme Eco31I) and 30 U of T4 DNA Ligase (Thermo, Fisher) in 2 µl 10x ligation buffer (10x ligation buffer was prepared by supplementing the Thermo, Fisher FastDigest buffer with 10mM dATP and 100mM DTT) to a final reaction volume of 20 µl with ddH2O. It is crucial for the reactions that the entry vectors are purified and diluted in pure water since buffer components might inhibit the Golden gate assembly. All reactions were incubated using the following cycling conditions: 2 min at 37 °C, 5 min at 20°C for 50 cycles, then 5 min at 50°C and 5 min at 80°C. 10µl of the reaction mix were used to transform chemical competent cells (Top10 strain from Life Technologies).

Publication protocol

Reactions were set up using 20 fmol of each Golden Gate entry vector and the destination vector, 10 U of BsaI Restriction Enzyme (Thermo, Fisher Fast Digest Enzyme Eco31I) and 30 U of T4 DNA Ligase (Thermo, Fisher) in 2 µl 10x ligation buffer (10x ligation buffer was prepared by supplementing the Thermo, Fisher FastDigest buffer with 10mM dATP and 100mM DTT) to a final reaction volume of 20 µl with ddH2O. It is crucial for the reactions that the entry vectors are purified and diluted in pure water since buffer components might inhibit the Golden gate assembly. All reactions were incubated using the following cycling conditions: 2 min at 37 °C, 5 min at 20°C for 50 cycles, then 5 min at 50°C and 5 min at 80°C. 10µl of the reaction mix were used to transform chemical competent cells (Top10 strain from Life Technologies).

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Manufacturer protocol

Download the product protocol from Thermo Fisher Scientific for FastDigest Eco31I (IIs class) below.

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