Restriction Enzymes BsaI / Eco31I

A restriction enzyme or restriction endonuclease is defined as a protein that recognizes a specific, short nucleotide sequence and cuts the DNA only at or near that site, known as restriction site or target sequence. The four most common types of restriction enzymes inclue: Type I (cleaves at sites remote from a recognition site), Type II (cleaves within or at short specific distances from a recognition site), Type III (cleave at sites a short distance from a recognition site), and Type IV (targets modified DNA- methylated, hydroxymethylated and glucosyl-hydroxymethylated DNA). The most common challenges with restriction digest include- 1. inactivation of enzyme, 2. incomplete or no digestion, and 3. unexpected cleavage. The enzyme should always be stored at -20C and multiple freeze-thaw cycles should be avoided in order to maintain optimal activity. Always use a control DNA digestion with the enzyme to ensure adequate activity (to avoid interference due to high glycerol in the enzyme). For complete digestion, make sure that the enzyme volume is 1/10th of the total reaction volume, optimal temperature is constantly maintained throughout the reaction, the total reaction time is appropriately calculated based on the amount of DNA to be digested, appropriate buffers should be used to ensure maximal enzymatic activity, and in case of a double digest, make sure that the two restriction sites are far enough so that the activity of one enzyme cannot interfere with activity of the other. Star activity (or off-target cleavage) and incomplete cleavage are potential challenges which may occur due to suboptimal enzymatic conditions or inappropriate enzyme storage. To avoid these, follow the recommended guidelines for storage and reactions, and always check for the efficacy of digestion along with purification of digested products on an agarose gel.

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Found 4 matching solutions for this experiment

Eco31I (BsaI) (10 U/µL)

Thermo Fisher Scientific

Protocol tips
The DNA assembly was carried out in 10 μl reaction comprised of ~50 ng Acceptor Vector and twice as many molar of the insert parts, in addition to 1 μl 1 mg/ml BSA (NEB), 1 μl T4 DNA ligase buffer (NEB or Thermo Fisher Scientific), 0.5 μl AarI (Thermo Fisher Scientific) for cloning in mUAV and Level 2 Acceptor Vectors or Eco31I (BsaI) (Thermo Fisher Scientific) for cloning in Level 1 Acceptor Vectors, and 0.5 μl T4 DNA ligase (NEB or Thermo Fisher Scientific). For reactions with AarI, extra 0.2 μl 50x oligos (0.025 mM) of the enzyme recognition sites were added. The one-tube reaction was incubated in a thermocycler for five times cycles of (37°C for 5 min, 16°C for 10 min) followed by 5 min digestion at 37°C and 5 min deactivation at 80°C. For the assembly of the 16 TU construct, the reaction was set in 20 μl with double amount of buffers and enzymes and the thermocycling conditions were altered to: 40 cycles of (37°C for 2.5 min, 16°C for 5 min) followed by 5 min digestion at 37°C and 5 min deactivation at 80°C. The sequences of all the primers and the resulting plasmids have been deposited to the public database Edinburgh Datashare (http://dx.doi.org/10.7488/ds/2261).
BsaI NEB#R0535

New England BioLabs

Protocol tips
For proof of principle modular assembly of ∼1 kb DNA from 4 fragments by MetClo, assembly reactions were set up using 60 fmol of each donor plasmid prepared from normal DH10B cells, 60 fmol of assembly vector prepared from DH10B strains stably expressing compatible methylase, 1,000 U T4 DNA ligase (NEB), and 5 U BsaI (NEB) or BpiI (Thermo Fisher Scientific) or 2.5 U LguI (Thermo Fisher Scientific) in 20 μl 1× T4 ligase buffer (NEB). The reaction condition was: 37°C 15 min, followed by 45 cycles of 37°C 2 min plus 16°C 5 min, then 37°C 20 min, and 80°C 5 min. Assembly reactions were transformed into normal DH10B chemical competent cells, and plated on LB plates with AIX selection (ampicillin 100 μg/ml, IPTG 100 μM, X-gal 50 μg/ml) at 37°C overnight. White colonies were expanded and screened by restriction digestion using the corresponding restriction enzyme and by DNA sequencing.
BsaI-HF®v2

New England BioLabs

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).
FastDigest Eco31I (IIs class)

Thermo Fisher Scientific

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).
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