Restriction Enzymes EcoRI

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 5 matching solutions for this experiment

FastDigest EcoRI

Thermo Fisher Scientific

Upstream tips	Protocol tips	Downstream tips
	The Fermentas FastDigestTM Restriction Enzyme EcoRI, which was specifically formulated to cleave DNA in just 5 min, was used in the experiment. The digestion media, which was prepared using 1 L of EcoRI (1 FDU/L) and 2 L enzyme reaction buffer (10 × FastDigestTM buffer), was placed on the dsDNA oligonucleotides immobilized microelectrode and incubated in a dark, humid environment for 5 min at 37 ◦C. Then the microelectrode surface was washed with Milli-Q water, dried under a stream of N2. Control probe DNA3 and complementary target DNA4 which were designed without specific recognition site of the EcoRI endonuclease were used under the same treatment. Enzyme reaction buffer was placed on the dsDNA immobilized microelectrode only as a control experiment.

Protocol tips
The Fermentas FastDigestTM Restriction Enzyme EcoRI, which was specifically formulated to cleave DNA in just 5 min, was used in the experiment. The digestion media, which was prepared using 1 L of EcoRI (1 FDU/L) and 2 L enzyme reaction buffer (10 × FastDigestTM buffer), was placed on the dsDNA oligonucleotides immobilized microelectrode and incubated in a dark, humid environment for 5 min at 37 ◦C. Then the microelectrode surface was washed with Milli-Q water, dried under a stream of N2. Control probe DNA3 and complementary target DNA4 which were designed without specific recognition site of the EcoRI endonuclease were used under the same treatment. Enzyme reaction buffer was placed on the dsDNA immobilized microelectrode only as a control experiment.

Protocol tips

The Fermentas FastDigestTM Restriction Enzyme EcoRI, which was specifically formulated to cleave DNA in just 5 min, was used in the experiment. The digestion media, which was prepared using 1 L of EcoRI (1 FDU/L) and 2 L enzyme reaction buffer (10 × FastDigestTM buffer), was placed on the dsDNA oligonucleotides immobilized microelectrode and incubated in a dark, humid environment for 5 min at 37 ◦C. Then the microelectrode surface was washed with Milli-Q water, dried under a stream of N2. Control probe DNA3 and complementary target DNA4 which were designed without specific recognition site of the EcoRI endonuclease were used under the same treatment. Enzyme reaction buffer was placed on the dsDNA immobilized microelectrode only as a control experiment.

Manufacturer protocol Publication protocol 1 Relevant paper

EcoRI-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

EcoRI NEB#R0101

New England BioLabs

Upstream tips	Protocol tips	Downstream tips
	Ten microliters of the reaction mixture was transferred to 10 μl of NEB 2 buffer with 5 U of MspI (NEB) and incubated for 3 hours at 37°C. Simultaneously, 0.5 μg of the same original DNA sample was incubated in NEB 2 buffer with 5 U of MspI (NEB) in a 10-μl reaction volume for 3 hours at 37°C. Next, 10 μl of EcoRI buffer (NEB) with 5 U of EcoRI (NEB) were added to 10-μl reactions (HpaII and MspI digestions) and 5 μl of 2.5× EcoRI buffer (NEB) with 5 U of EcoRI (NEB) were added to a 20-μl HpaII + MspI reaction volume followed by incubation for an additional 1.5 hours at 37°C.

Protocol tips
Ten microliters of the reaction mixture was transferred to 10 μl of NEB 2 buffer with 5 U of MspI (NEB) and incubated for 3 hours at 37°C. Simultaneously, 0.5 μg of the same original DNA sample was incubated in NEB 2 buffer with 5 U of MspI (NEB) in a 10-μl reaction volume for 3 hours at 37°C. Next, 10 μl of EcoRI buffer (NEB) with 5 U of EcoRI (NEB) were added to 10-μl reactions (HpaII and MspI digestions) and 5 μl of 2.5× EcoRI buffer (NEB) with 5 U of EcoRI (NEB) were added to a 20-μl HpaII + MspI reaction volume followed by incubation for an additional 1.5 hours at 37°C.

Protocol tips

Ten microliters of the reaction mixture was transferred to 10 μl of NEB 2 buffer with 5 U of MspI (NEB) and incubated for 3 hours at 37°C. Simultaneously, 0.5 μg of the same original DNA sample was incubated in NEB 2 buffer with 5 U of MspI (NEB) in a 10-μl reaction volume for 3 hours at 37°C. Next, 10 μl of EcoRI buffer (NEB) with 5 U of EcoRI (NEB) were added to 10-μl reactions (HpaII and MspI digestions) and 5 μl of 2.5× EcoRI buffer (NEB) with 5 U of EcoRI (NEB) were added to a 20-μl HpaII + MspI reaction volume followed by incubation for an additional 1.5 hours at 37°C.

Publication protocol 1 Relevant paper

EcoRI restriction enzyme

Takara Bio Inc

Upstream tips	Protocol tips	Downstream tips
	The 5′ PpAOS1 genomic fragment was digested with XbaI (Takara Bio Inc., Shiga, Japan) and EcoRI (Takara Bio Inc., Shiga, Japan) and inserted into pTN182, which carries a G418-resistant cassette, digested with XbaI (Takara Bio Inc., Shiga, Japan) and EcoRI (Takara Bio Inc., Shiga, Japan) to obtain pTN182-PpAOS1KO5′.

Protocol tips
The 5′ PpAOS1 genomic fragment was digested with XbaI (Takara Bio Inc., Shiga, Japan) and EcoRI (Takara Bio Inc., Shiga, Japan) and inserted into pTN182, which carries a G418-resistant cassette, digested with XbaI (Takara Bio Inc., Shiga, Japan) and EcoRI (Takara Bio Inc., Shiga, Japan) to obtain pTN182-PpAOS1KO5′.

Manufacturer protocol Publication protocol 1 Relevant paper

EcoRI R6011

Promega

Upstream tips	Protocol tips	Downstream tips
	The clone 4G4E4.0 encompassing a 4-kb EcoRI 59 fragment of the human huntingtin gene (including exon 1 and upstream noncoding region) (Lin et al., 1995) was characterized by restriction endonuclease mapping with EcoRI, HindIII, and SfiI.

Protocol tips
The clone 4G4E4.0 encompassing a 4-kb EcoRI 59 fragment of the human huntingtin gene (including exon 1 and upstream noncoding region) (Lin et al., 1995) was characterized by restriction endonuclease mapping with EcoRI, HindIII, and SfiI.

Manufacturer protocol Publication protocol 1 Relevant paper

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