Restriction Enzymes NcoI

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 include: 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 the 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, the 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 the 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 6 matching solutions for this experiment

NcoI-HF®

New England BioLabs

Upstream tips	Protocol tips	Downstream tips
	All enzymes used (BamHI, EcoRI, NcoI, SmaI, XbaI, and XhoI) were from New England Biolabs and had a concentration of 20 000 U/mL. The high-fidelity versions of the enzymes BamHI, EcoRI, and NcoI were employed in the experiments. Single-enzyme digestions were conducted by inserting 1 μL of enzyme for every 10 μL of sample and gently mixing via repeated pipetting. Multienzyme digestions were conducted using the same DNA-to-enzyme ratio for each individual enzyme, with 1 μL of each enzyme per 10 μL volume of sample, i.e., three enzyme digest will contain 3 μL of enzyme solution per 10 μL of total solution volume. The solutions then sat out in a room-temperature environment for a minimum of 1 h prior to analysis.

Protocol tips
All enzymes used (BamHI, EcoRI, NcoI, SmaI, XbaI, and XhoI) were from New England Biolabs and had a concentration of 20 000 U/mL. The high-fidelity versions of the enzymes BamHI, EcoRI, and NcoI were employed in the experiments. Single-enzyme digestions were conducted by inserting 1 μL of enzyme for every 10 μL of sample and gently mixing via repeated pipetting. Multienzyme digestions were conducted using the same DNA-to-enzyme ratio for each individual enzyme, with 1 μL of each enzyme per 10 μL volume of sample, i.e., three enzyme digest will contain 3 μL of enzyme solution per 10 μL of total solution volume. The solutions then sat out in a room-temperature environment for a minimum of 1 h prior to analysis.

Protocol tips

All enzymes used (BamHI, EcoRI, NcoI, SmaI, XbaI, and XhoI) were from New England Biolabs and had a concentration of 20 000 U/mL. The high-fidelity versions of the enzymes BamHI, EcoRI, and NcoI were employed in the experiments. Single-enzyme digestions were conducted by inserting 1 μL of enzyme for every 10 μL of sample and gently mixing via repeated pipetting. Multienzyme digestions were conducted using the same DNA-to-enzyme ratio for each individual enzyme, with 1 μL of each enzyme per 10 μL volume of sample, i.e., three enzyme digest will contain 3 μL of enzyme solution per 10 μL of total solution volume. The solutions then sat out in a room-temperature environment for a minimum of 1 h prior to analysis.

Publication protocol 1 Relevant paper

FastDigest NcoI

Thermo Fisher Scientific

Upstream tips	Protocol tips	Downstream tips
	We digest the amplicon and pBAD-Ara::cmyc-His with FastDigest NcoI and FastDigest BgIII (both ThermoFischer Scientific) for 30min in Fast Digest buffer according to the supplier’s double digestion protocol. We treated the vector with alkaline phosphatase (NEB) at 37°C for 1h to avoid recircularization. A 1:5 vector:insert was incubated with T4 DNA Ligase (ThermoFischer Scientific) at 22°C O/N. The reaction product was dialysed to MiliQ water for 1h using dialysis disks.

Protocol tips
We digest the amplicon and pBAD-Ara::cmyc-His with FastDigest NcoI and FastDigest BgIII (both ThermoFischer Scientific) for 30min in Fast Digest buffer according to the supplier’s double digestion protocol. We treated the vector with alkaline phosphatase (NEB) at 37°C for 1h to avoid recircularization. A 1:5 vector:insert was incubated with T4 DNA Ligase (ThermoFischer Scientific) at 22°C O/N. The reaction product was dialysed to MiliQ water for 1h using dialysis disks.

Protocol tips

We digest the amplicon and pBAD-Ara::cmyc-His with FastDigest NcoI and FastDigest BgIII (both ThermoFischer Scientific) for 30min in Fast Digest buffer according to the supplier’s double digestion protocol. We treated the vector with alkaline phosphatase (NEB) at 37°C for 1h to avoid recircularization. A 1:5 vector:insert was incubated with T4 DNA Ligase (ThermoFischer Scientific) at 22°C O/N. The reaction product was dialysed to MiliQ water for 1h using dialysis disks.

Manufacturer protocol Publication protocol 1 Relevant paper

NcoI NEB#R0193

New England BioLabs

Upstream tips	Protocol tips	Downstream tips
	These three fragments were connected with pUC19, generating plasmid pUC-fdnG, and then digested with NcoI (NEB) and transformed into the electrocompetent G. sulfurreducens strain PCA-∆pilB∆hybL. All mutants were verified by PCR (Supplementary Fig. S1) and Sanger sequencing.

Protocol tips
These three fragments were connected with pUC19, generating plasmid pUC-fdnG, and then digested with NcoI (NEB) and transformed into the electrocompetent G. sulfurreducens strain PCA-∆pilB∆hybL. All mutants were verified by PCR (Supplementary Fig. S1) and Sanger sequencing.

Publication protocol 1 Relevant paper

NcoI restriction enzyme

Takara Bio Inc

Upstream tips	Protocol tips	Downstream tips
	Plasmid pGFPuv4-NF, which contains a non-fluorescent GFP gene coding sequence, was prepared by restriction digestion with NcoI (Takara) followed by treatment of pGFPuv4 (Ito et al., 1999) with T4 DNA polymerase (Takara) and re-ligation with T4 DNA ligase (Takara).

Protocol tips
Plasmid pGFPuv4-NF, which contains a non-fluorescent GFP gene coding sequence, was prepared by restriction digestion with NcoI (Takara) followed by treatment of pGFPuv4 (Ito et al., 1999) with T4 DNA polymerase (Takara) and re-ligation with T4 DNA ligase (Takara).

Manufacturer protocol Publication protocol 1 Relevant paper

NcoI (10 U/µL)

Thermo Fisher Scientific

Upstream tips	Protocol tips	Downstream tips
	The synthetic gene, provided in a standard cloning vector, was subcloned into a pET28 plasmid (Novagen, Merck Millipore, Burlington, Massachusetts, USA) by restriction cloning using NcoI and XhoI (Fermentas, Waltham, Massachusetts, USA) as restriction enzymes.

Protocol tips
The synthetic gene, provided in a standard cloning vector, was subcloned into a pET28 plasmid (Novagen, Merck Millipore, Burlington, Massachusetts, USA) by restriction cloning using NcoI and XhoI (Fermentas, Waltham, Massachusetts, USA) as restriction enzymes.

Manufacturer protocol Publication protocol 1 Relevant paper

NcoI R6513

Promega

Upstream tips	Protocol tips	Downstream tips
	PCR products were digested with NcoI (Promega) overnight at 37°C. PCR products and restriction enzyme (RE) digests were visualized on ethidium bromide-stained 10% polyacrylamide gels.

Protocol tips
PCR products were digested with NcoI (Promega) overnight at 37°C. PCR products and restriction enzyme (RE) digests were visualized on ethidium bromide-stained 10% polyacrylamide gels.

Manufacturer protocol Publication protocol 1 Relevant paper

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