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Troubleshooting for restriction endonuclease digestion

Troubleshooting for restriction endonuclease digestion

The followings in the table are possible explanations and solutions of each problem that is commonly

encountered during restriction endonuclease digestion.

 

Trouble : DNA substrates are not cleaved.

Cause

Explanation and solution

Impure DNA

l Substrate DNA of low purity is difficult to be digested.

l Remove proteins or other contaminants with phenol/chloroform extraction and ethanol precipitation, or by using a DNA purification kit. Digest the purified DNA with restriction endonucleases.

l Elevate MgCl2 up to 50 mM when digesting low purity DNA.

Presence of inhibitors.

l Restriction endonucleases are inhibited by SDS, phenol, EDTA, chloroform, ethanol, CsCl, high salt, and additives in microcentrifuge tubes

l Most of the inhibitors can be removed by phenol/chloroform treatment and ethanol precipitation.

l Use commercially available DNA clean-up kits to remove inhibitors.

Methylation of DNA

(e.g., dam, dcm)

l Confirm the influence of methylation on restriction endonucleases prior to use.

l Amplify substrate plasmid DNA in host cells which do not contain methylase (e.g., M110; dam- dcm-) to avoid methylation.

l Use an isoschizomers that is not affected by methylation.

DNA is not methylated

l Some restriction endonucleases such as Dpn I (Cat.# R054) require a methylated base (N6– methylated adenine) for their activity.

Inactive restriction

enzyme

l Confirm the unit activity of the restriction endonucleases by digesting either bacteriophage lambda DNA or other DNA substrates.

l Use a new batch of restriction endonuclease.

Unsuitable reaction

conditions

l Refer to proper reaction conditions from suppliers.

Wrong DNA sequence

l Confirm the nucleotide sequences of substrate DNA and the number of restriction enzyme recognition sites.

 

Trouble : DNA substrates are partially cleaved.

Cause

Explanation and solution

Substrate DNA is not pure

l Remove proteins or other contaminants with phenol/chloroform extraction and ethanol precipitation, or by using commercially available DNA clean-up kits Digest the purified DNA with restriction endonucleases.

Partial loss or inactivation

of the restriction

endonucleases activity

l Reaction conditions may not be optimal for one of the restriction endonucleases, when cleaving DNA with two or more restriction endonucleases simultaneously.

l In general, one unit of a restriction endonuclease is defined as the amount of enzyme required to completely cleave 1 μg of linear DNA within an hour. However, supercoiled DNA often requires more restriction endonucleases. Therefore, higher concentrations of restriction endonucleases should be used to cleave supercoiled DNA.

l Check whether a restriction endonuclease has partial activity by using a standard substrate. The simplest alternative way to determine activity of a restriction endonuclease is to cleave a substrate DNA with a known number of restriction sites.

Presence of inhibitors

l Restriction endonucleases are inhibited by SDS, phenol, EDTA, chloroform, ethanol, CsCl, high salt, and additives in microcentrifuge tubes.

l Restriction endonucleases can be inhibited by the presence of over 5% of glycerol in the reaction mixture. Restriction endonucleases should not be over 10% of the total reaction volume.

l Most of the inhibitors can be removed by phenol/chloroform treatment and ethanol precipitation.

l Use commercially available DNA clean-up kits to remove inhibitors.

Pipetting error

l While pipetting small quantity of highly viscous solutions such as concentrated DNA or enzyme storage buffers, the actual quantity take may be more or less than required.

l Use correct amount of enzyme.

Incomplete mixing

l Add restriction endonucleases last to the reaction mixture and mix thoroughly.

l Use a vortex, but gentle mixing is recommended.

Loss of enzyme activity

during dilution prior to use

l Do not dilute restriction endonucleases unless it is necessary.

l If dilution is required, dilute in the storage buffer which contains BSA (0.1 mg/ml) or in diluent buffer recommended by suppliers, and mix gently prior to use.

l Avoid dilution in water.

l It is not suitable to store enzymes in dilution buffer for a long time, because activity may be lost rapidly.

High or low reaction

temperature.

l Suboptimal reaction temperatures result in partial activity of restriction endonucleases

l Carry out the reaction at the temperature recommended for each restriction endonuclease.

Reannealing of

complementary DNA ends

l The two cos ends of bacteriophage λDNA can be annealed during restriction endonuclease digestion, joining two fragments into one. It appears as if incomplete reaction occurred. Incubation at 65 for 5 min before gel electrophoresis can separate the cos-bearing DNA fragments.

l Denaturation of small DNA fragments can be prevented by adding more salt to a restriction endonuclease buffer, which increases Tm of duplex DNA.

Denaturation of restriction

endonucleases

l Most of the restriction endonucleases are inactivated by heat treatment. In addition, restriction endonucleases can be denatured like most other proteins by oxidation when exposed to air .

l Gently mix the reaction mixture containing restriction endonucleases to minimize their contact with air.

Supercoiled substrate

DNA

l In comparison to linear DNA, supercoiled DNA requires more units of restriction endonucleases for complete cleavage. In many cases, one unit of restriction endonucleases is determined based on cleavage of linear DNA such as bacteriophage λDNA. Therefore, use of increased units of restriction endonucleases solves the problem.

l If more than two enzymes are used, cleave first with a restriction endonuclease which is not affected by supercoiled structure and then add the second enzyme for complete digestion.

l Supercoiled DNA can be efficiently cleaved after treating with topoisomerase to relax the supercoiled structure.

Substrates with many

restriction enzyme

cleavage sites.

l The number of cleavage sites can affect the amount of restriction endonucleases for complete digestion. Increase the amount of restriction enzymes when DNA to be cleaved has markly more sites than that of the standard DNA.            

 

Trouble : Smeared DNA fragments are observed after restriction endonuclease digestion.

Cause

Explanation and solution

Nuclease contamination

in the reagents

l Bands of DNA fragments disappear completely if the reagents and substrate DNA used are heavily contaminated with non-specifific nucleases. Digested bands look smeared with less degree of nuclease contamination.

l Check each reagent for the presence of contaminated nuclease, and then use a new reagent.

Inappropriate reaction

conditions

l Although it is rare in commercially available restriction endonucleases, a nuclease present in a restriction enzyme or in other reagents can be activated in inappropriate reaction conditions, degrading substrate DNA.

l Refer to proper reaction conditions from suppliers.

Inappropriate storage or

handling of restriction

endonucleases.

l Inappropriate storage can cause selective inactivation of restriction endonucleases. This makes a nuclease, if present in the restriction endonucleases, become the major activity.

l Take out the restriction endonucleases just prior to use, and place it back in freezer as soon as possible.

l Use a cold enzyme-carrying rack to keep the temperature low.

l Mix gently to prevent formation of bubbles that may cause inactivation of restriction endonucleases.

Storage of enzymes in

diluted conditions.

l Storage of restriction endonucleases in diluted conditions can also cause selective inactivation of restriction endonucleases, making a nuclease, if present in the restriction endonucleases, become the major activity.

l Avoid direct dilution in water.

l Dilute in BSA-containing 1X reaction buffer solution just prior to use. Use the diluted enzyme as soon as possible after dilution.

l It is best to store restriction endonucleases under the conditions recommended by suppliers.

 

Trouble : Additional DNA fragments are observed.

Cause

Explanation and solution

Star activity

l Some restriction endonucleases can cleave other sites that contain sequences similar to but not identical with their recognition sequence. This is called star activity and occurs under suboptimal reaction conditions.

l Common causes of star activity are excess use of restriction endonucleases (>100 units/μg), glycerol concentration over 5%, divalent metal ions including manganese (in place of magnesium), nonoptimal concentrations of NaCl, and alkaline pH (>pH 8.0) of reaction buffer.

l Organic solvents including DMSO and ethanol are also principal factors that cause star activity.

l Use pure DNA preparation which does not contain excess salt or organic solvents.

l Use reaction conditions recommended by the suppliers.

Contaminated with unwanted DNA.

l If unexpected bands appear after cleaving DNA with known cleavage sites for a specific restriction endonuclease, it is likely that substrate DNA is contaminated with DNA other than the substrate.

l Confirm if reagents such as reaction or agarose loading buffers are contaminated with DNA

l Use a new batch of DNA-free reagent.

Use of wrong restriction

endonucleases.

l If additional DNA bands from bacteriophage l DNA or other substrate DNA that contains well-defined restriction sites are observed, it is likely that either excess restriction endonucleases or another restriction endonuclease is used.

l Reduce the amount of enzyme used.

l Use the right restriction endonucleases.

Reduction of reaction mixture volumes due to prolonged incubation.

l Prolonged incubation at high temperatures can lead to evaporation of reaction mixtures, thus increasing the concentration of enzyme, glycerol, salt and other components that cause star activity.

l Use mineral oil to prevent evaporation

l Reduce reaction time by increasing the amount of enzyme.

Wrong nucleotide sequences

l Confirm the recognition sequence and number of restriction endonuclease sites.

 

Trouble : DNA fragments are not detected after digestion

Cause

Explanation and solution

Inaccurate concentrations of

substrate DNA

l Confirm concentration of substrate DNA by agarose gel electrophoresis.

l Contamination with high levels of RNA or salt such as guanidine results in false measurement of DNA concentrations with 260 nm absorbance.

l If necessary, treat DNA preparation with RNase or remove salt by ethanol precipitation.

Nuclease contamination

in the substrate DNA

l DNA can be severely contaminated with endonucleases when DNA is isolated from endA(+) host strains.

l The contaminating endoucleases degrade substrate DNA in buffer for most restriction endonucleases.

l To avoid this problem, use endA(-) host bacteria when amplifying plasmid DNA for restriction endonuclease cleavage.

l Use DNA purification system that can remove endonucleases.

Nuclease contamination

in the reagents

l Use new chemicals, after confirmation of nuclease contamination in each reagent.

l Growth of bacteria and mold in a reagent is a potential source of nuclease contamination.


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