Support, restriction enzymes: Fermentas Guide for Successful Digestions

Restriction Enzymes

Modifying Enzymes

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Fermentas Guide for Successful Digestions

FAQs about Restriction Enzymes
Problems:

No digestion or incompletely digested DNA

Atypical cleavage pattern

Diffused DNA zones, gel shift

Low efficiency of restriction fragment ligation

Possible Cause and Recommended Solution:

Problem 1. No digestion or incompletely digested DNA

Possible Cause Recommended Solution

The DNA sample contains impurities that inhibit restriction enzymes. To check if these contaminants inhibit restriction enzymes, digest the control DNA. In parallel, digest your sample supplemented with the control DNA.

If PCR products are used directly after amplification, dilute the sample 3-fold with the recommended buffer prior digestion (see Cleavage of PCR Products Directly After Amplification).

If DNA is purified using silica or resin suspensions, re-centrifuge your sample (10 min at 10,000 rpm) to remove any remnant particles.

Consider re-purification of the sample DNA.

Some restriction enzymes cleave supercoiled plasmid DNA with lower efficiency. Add more of the restriction enzyme (5-10 u of restriction enzyme per 1 µg of DNA).

The DNA sequence context influences the efficiency of digestion. Therefore, some DNA sites are cleaved slowly, or they are not cleaved at all (see Site Preferences by Restriction Enzymes).

Add 5-10 u of restriction enzymes per 1 µg of DNA.

Try another isoschizomer (see Table "Alphabetic List of Commercially Available Restriction Enzymes")

Some restriction enzymes, like AarI, BveI, Cfr10I, Eam1104, Eco57I, EcoRII, SfiI, require at least two target sites per DNA molecule for efficient cleavage (see Site Preferences by Restriction Enzymes).

Evaluate the number of recognition sites per DNA molecule.

If there is only one recognition site per DNA molecule, add an activator DNA containing the same enzyme-specific recognition site (Fermentas restriction enzymes such as AarI and BveI are supplied with activating oligonucleotides).

Some restriction enzymes cleave DNA poorly, if the recognition site is too close to the end of the DNA molecule.

Refer to Table "Cleavage of Restriction Targets Located in Close Vicinity within pUC19 MCS" and Table "Cleavage Efficiency Close to the Termini of PCR Fragments" to check the effectiveness of restriction enzymes at the ends of DNA.

Consider direct cloning of your PCR product into Fermentas vectors for blunt and TA cloning (CloneJET™ PCR Cloning Kit or InsTAclone™ PCR Cloning Kit*).
* Available in certain countries only.

The recognition site for the restriction enzyme is not present in the DNA molecule. Re-check the DNA sequence and cloning strategy.

The DNA molecule is methylated at the site which is recognized by a methylation-sensitive restriction enzyme.
Note
PCR products are NOT methylated when the PCR is carried out with standard dNTPs and non-methylated primers.

Identify which type of DNA methylation can occur (see Digestion of Methylated DNA).

Use Tables in Digestion of Methylated DNA to check if methylation could influence DNA digestion.

If methylation is the reason for impaired DNA cleavage, we suggest the following:

propagate your plasmid in E.coli dam-, dcm- strain. (E.coli GM2163 dam-, dcm-, strain is available for free upon request under #M0099),

use the REsearch™ engine or check the Fermentas catalog for availability of a restriction enzyme isoschizomer which is not sensitive to DNA methylation.

Restriction enzyme DpnI was used to digest DNA containing unmethylated targets.

If the cleavage site is not important for your experiment use other neoschizomer: Bsp143I or MboI.

If the cleavage site must be retained, propagate your plasmid in E.coli dam+ strains.

Suboptimal reaction conditions.

Digest your DNA under the specific conditions indicated in the product’s Certificate of Analysis (supplied with each enzyme).

Use the Fermentas buffer supplied with the restriction enzyme.

Use additives where required.

Perform digestion at the optimal temperature; refer Table "Activity of Mesophilic and Thermophilic Enzymes at 37°C" for data on the activity of thermophilic enzymes at 37°C.

Ensure the volume of the reaction mixture was not reduced due to evaporation during incubation; the resulting increase in salt concentration may reduce enzyme activity.

The restriction enzyme has been diluted improperly.

Never dilute enzyme in water or 10X reaction buffer.

Avoid dilution in 1X reaction buffer in the absence of DNA.

Dilute restriction enzymes with Fermentas Dilution Buffer. Restriction enzymes diluted with this buffer are stable for at least 3-4 weeks at -20°C (for more information see).

The restriction enzyme was added to a reaction buffer with low ionic strength in the absence of stabilizing agents. The restriction enzyme should always be the last component added to the reaction mixture.

The glycerol concentration in the reaction mixture is too high.

The glycerol concentration in the reaction mixture should not exceed 5%. Thus, the volume of the restriction enzyme added to the mixture should not exceed 1/10 of the total volume.

Alw21I, BpiI, Bsp68I, BspTI, Eco32I, Eco91I, EcoRI, Hin6I, HinfI, Mph1103I, Mva1269I and NcoI are especially sensitive to the high glycerol concentration in the reaction mixture.

The DNA concentration in the reaction mixture is too high or too low The optimal range of DNA concentration in the reaction mixture is 0.02-0.1 µg/µl.

The restriction enzyme has been inactivated due to improper storage or handling.

Check the expiration date.

Check if the enzyme has been stored at -20°C.

Perform a digestion of control DNA.

Problem 2. Atypical cleavage pattern

Possible Cause Recommended Solution

Incomplete digestion of DNA (see Considerations for Partial Digestion). Add more enzyme or prolong the incubation.

Star (relaxed) activity of restriction enzyme (see Star Activity).

Add less restriction enzyme (not more than 10 u of restriction enzyme per 1 µg DNA).

Digest DNA in the recommended buffer.

Ensure that the glycerol concentration in the reaction mixture does not exceed 5%.

Shorten the incubation time.

Ensure the volume of the reaction mixture was not reduced due to evaporation during incubation; the resulting increase in glycerol concentration may cause star activity.

Some newly generated target sites in constructed DNA were overseen. Recheck your DNA sequence and cloning strategy to identify all target sites.

If an atypical pattern of DNA digestion persists, the restriction enzyme or buffer could be contaminated with another restriction enzyme due to improper handling. Use a new tube of enzyme or/and buffer.

The sample DNA preparation is a mixture of two different DNAs. Prepare non-contaminated DNA.

Problem 3. Diffused DNA zones, gel shift

Possible Cause Recommended Solution

Contaminated substrate.

Purify the DNA sample by phenol/chloroform extraction and ethanol precipitation (see Protocol for DNA Purification after Enzymatic Reaction by Phenol/chloroform Extraction and Alcohol Precipitation).

Perform two control reactions: one without a restriction enzyme and one with another restriction enzyme.

The enzyme was contaminated due to improper handling.

Use a new tube of enzyme.

Verify enzyme activity with the substrate indicated in the product’s Certificate of Analysis.

Bacterial growth within the buffer(s).

Use a new tube of buffer.

Store all buffers at -20°C.

Protein binding to the substrate DNA affects the electrophoretic mobility of digestion products (gel shift). Restriction enzymes AarI, AloI, BdaI, BspPI, EcoRII, Eco57I, GsuI, TauI, TsoI are particularly prone to bind their substrate DNA. Use the 6X DNA Loading Dye & SDS Solution for sample preparation or heat the digested DNA in the presence of SDS prior to electrophoresis.

Problem 4. Low efficiency of restriction fragment ligation

Possible Cause Recommended Solution

Restriction enzyme is still active in the ligation mixture.

Check the thermostability of the restriction enzyme in the product description, Certificate of Analysis or Table "Reaction Conditions for Restriction Enzymes".

Purify the digested DNA by phenol/chloroform extraction and ethanol precipitation.

Restriction enzyme did not cut target sites situated close to the DNA termini.

Refer to Table "Cleavage of Restriction Targets Located in Close Vicinity within pUC19 MCS" and Table "Cleavage Efficiency Close to the Termini of PCR Fragments" to check the effectiveness of restriction enzymes at the ends of DNA.

Consider direct cloning of your PCR product into Fermentas vectors for blunt and TA cloning (CloneJET™ PCR Cloning Kit or InsTAclone™ PCR Cloning Kit*).
* Available in certain countries only.

Restriction fragments with blunt ends are more difficult to ligate.

Use 100-500 u/ml of ligase for ligation (final concentration).

Add 10% of polyethylene glycol (supplied with ligase) to the reaction mixture.

catalog@fermentas.com

Updated sausio 31, 2008 16:23

Possible Cause	Recommended Solution
The DNA sample contains impurities that inhibit restriction enzymes.	To check if these contaminants inhibit restriction enzymes, digest the control DNA. In parallel, digest your sample supplemented with the control DNA. If PCR products are used directly after amplification, dilute the sample 3-fold with the recommended buffer prior digestion (see Cleavage of PCR Products Directly After Amplification). If DNA is purified using silica or resin suspensions, re-centrifuge your sample (10 min at 10,000 rpm) to remove any remnant particles. Consider re-purification of the sample DNA.
Some restriction enzymes cleave supercoiled plasmid DNA with lower efficiency.	Add more of the restriction enzyme (5-10 u of restriction enzyme per 1 µg of DNA).
The DNA sequence context influences the efficiency of digestion. Therefore, some DNA sites are cleaved slowly, or they are not cleaved at all (see Site Preferences by Restriction Enzymes).	Add 5-10 u of restriction enzymes per 1 µg of DNA. Try another isoschizomer (see Table "Alphabetic List of Commercially Available Restriction Enzymes")
Some restriction enzymes, like AarI, BveI, Cfr10I, Eam1104, Eco57I, EcoRII, SfiI, require at least two target sites per DNA molecule for efficient cleavage (see Site Preferences by Restriction Enzymes).	Evaluate the number of recognition sites per DNA molecule. If there is only one recognition site per DNA molecule, add an activator DNA containing the same enzyme-specific recognition site (Fermentas restriction enzymes such as AarI and BveI are supplied with activating oligonucleotides).
Some restriction enzymes cleave DNA poorly, if the recognition site is too close to the end of the DNA molecule.	Refer to Table "Cleavage of Restriction Targets Located in Close Vicinity within pUC19 MCS" and Table "Cleavage Efficiency Close to the Termini of PCR Fragments" to check the effectiveness of restriction enzymes at the ends of DNA. Consider direct cloning of your PCR product into Fermentas vectors for blunt and TA cloning (CloneJET™ PCR Cloning Kit or InsTAclone™ PCR Cloning Kit). Available in certain countries only.
The recognition site for the restriction enzyme is not present in the DNA molecule.	Re-check the DNA sequence and cloning strategy.
The DNA molecule is methylated at the site which is recognized by a methylation-sensitive restriction enzyme. Note PCR products are NOT methylated when the PCR is carried out with standard dNTPs and non-methylated primers.	Identify which type of DNA methylation can occur (see Digestion of Methylated DNA). Use Tables in Digestion of Methylated DNA to check if methylation could influence DNA digestion. If methylation is the reason for impaired DNA cleavage, we suggest the following: propagate your plasmid in E.coli dam-, dcm- strain. (E.coli GM2163 dam-, dcm-, strain is available for free upon request under #M0099), use the REsearch™ engine or check the Fermentas catalog for availability of a restriction enzyme isoschizomer which is not sensitive to DNA methylation.
Restriction enzyme DpnI was used to digest DNA containing unmethylated targets.	If the cleavage site is not important for your experiment use other neoschizomer: Bsp143I or MboI. If the cleavage site must be retained, propagate your plasmid in E.coli dam+ strains.
Suboptimal reaction conditions.	Digest your DNA under the specific conditions indicated in the product’s Certificate of Analysis (supplied with each enzyme). Use the Fermentas buffer supplied with the restriction enzyme. Use additives where required. Perform digestion at the optimal temperature; refer Table "Activity of Mesophilic and Thermophilic Enzymes at 37°C" for data on the activity of thermophilic enzymes at 37°C. Ensure the volume of the reaction mixture was not reduced due to evaporation during incubation; the resulting increase in salt concentration may reduce enzyme activity.
The restriction enzyme has been diluted improperly.	Never dilute enzyme in water or 10X reaction buffer. Avoid dilution in 1X reaction buffer in the absence of DNA. Dilute restriction enzymes with Fermentas Dilution Buffer. Restriction enzymes diluted with this buffer are stable for at least 3-4 weeks at -20°C (for more information see).
The restriction enzyme was added to a reaction buffer with low ionic strength in the absence of stabilizing agents.	The restriction enzyme should always be the last component added to the reaction mixture.
The glycerol concentration in the reaction mixture is too high.	The glycerol concentration in the reaction mixture should not exceed 5%. Thus, the volume of the restriction enzyme added to the mixture should not exceed 1/10 of the total volume. Alw21I, BpiI, Bsp68I, BspTI, Eco32I, Eco91I, EcoRI, Hin6I, HinfI, Mph1103I, Mva1269I and NcoI are especially sensitive to the high glycerol concentration in the reaction mixture.
The DNA concentration in the reaction mixture is too high or too low	The optimal range of DNA concentration in the reaction mixture is 0.02-0.1 µg/µl.
The restriction enzyme has been inactivated due to improper storage or handling.	Check the expiration date. Check if the enzyme has been stored at -20°C. Perform a digestion of control DNA.

Possible Cause	Recommended Solution
Incomplete digestion of DNA (see Considerations for Partial Digestion).	Add more enzyme or prolong the incubation.
Star (relaxed) activity of restriction enzyme (see Star Activity).	Add less restriction enzyme (not more than 10 u of restriction enzyme per 1 µg DNA). Digest DNA in the recommended buffer. Ensure that the glycerol concentration in the reaction mixture does not exceed 5%. Shorten the incubation time. Ensure the volume of the reaction mixture was not reduced due to evaporation during incubation; the resulting increase in glycerol concentration may cause star activity.
Some newly generated target sites in constructed DNA were overseen.	Recheck your DNA sequence and cloning strategy to identify all target sites.
If an atypical pattern of DNA digestion persists, the restriction enzyme or buffer could be contaminated with another restriction enzyme due to improper handling.	Use a new tube of enzyme or/and buffer.
The sample DNA preparation is a mixture of two different DNAs.	Prepare non-contaminated DNA.

Possible Cause	Recommended Solution
Contaminated substrate.	Purify the DNA sample by phenol/chloroform extraction and ethanol precipitation (see Protocol for DNA Purification after Enzymatic Reaction by Phenol/chloroform Extraction and Alcohol Precipitation). Perform two control reactions: one without a restriction enzyme and one with another restriction enzyme.
The enzyme was contaminated due to improper handling.	Use a new tube of enzyme. Verify enzyme activity with the substrate indicated in the product’s Certificate of Analysis.
Bacterial growth within the buffer(s).	Use a new tube of buffer. Store all buffers at -20°C.
Protein binding to the substrate DNA affects the electrophoretic mobility of digestion products (gel shift). Restriction enzymes AarI, AloI, BdaI, BspPI, EcoRII, Eco57I, GsuI, TauI, TsoI are particularly prone to bind their substrate DNA.	Use the 6X DNA Loading Dye & SDS Solution for sample preparation or heat the digested DNA in the presence of SDS prior to electrophoresis.

Possible Cause	Recommended Solution
Restriction enzyme is still active in the ligation mixture.	Check the thermostability of the restriction enzyme in the product description, Certificate of Analysis or Table "Reaction Conditions for Restriction Enzymes". Purify the digested DNA by phenol/chloroform extraction and ethanol precipitation.
Restriction enzyme did not cut target sites situated close to the DNA termini.	Refer to Table "Cleavage of Restriction Targets Located in Close Vicinity within pUC19 MCS" and Table "Cleavage Efficiency Close to the Termini of PCR Fragments" to check the effectiveness of restriction enzymes at the ends of DNA. Consider direct cloning of your PCR product into Fermentas vectors for blunt and TA cloning (CloneJET™ PCR Cloning Kit or InsTAclone™ PCR Cloning Kit). Available in certain countries only.
Restriction fragments with blunt ends are more difficult to ligate.	Use 100-500 u/ml of ligase for ligation (final concentration). Add 10% of polyethylene glycol (supplied with ligase) to the reaction mixture.