Can I double digest my DNA using a Thermo Scientific conventional restriction enzyme and a FastDigest restriction enzyme?
For optimal results with fast reaction and 100% buffer compatibility, we highly recommend using FastDigest restriction enzymes in double digestion. In certain cases however, it may be possible to perform double digestion using a mix of Thermo Scientific conventional and Fastdigest restriction enzymes. For specific recommendations, please contact our technical service with detailed information about the enzymes and DNA template you plan to use.
Why do you recommend only 2 µL of 10X Reaction Buffer when digesting unpurified PCR product in a 30 µL reaction?
We recommend only 2 µl 10X Buffer in digestion of unpurified PCR products in 30 ul since salts and ions from the PCR reaction would be carried over to the digestion reaction.
Find additional tips, troubleshooting help, and resources within our Restriction Enzyme Cloning Support Center.
What are key factors promoting star activity?
Star activty may be contributed by:
• Prolonged incubation
• High enzyme concentration
• High glycerol concentration (usually 5% or higher)
• Small reaction volume
Find additional tips, troubleshooting help, and resources within our Restriction Enzyme Cloning Support Center.
Unexpected DNA bands were observed on agarose gel electrophoresis after restriction digestion. What may have caused this?
Unexpected cleavage patterns may be caused by the following reasons:
• Star activity of the restriction enzyme: Make sure to follow the reaction recommendations as specified in the protocol. Star activity may be improved by changing several key factors such as decreasing the reaction time, increasing the reaction volume, and decreasing the enzyme amount.
• Partial or incomplete cleavage (incomplete restriction reaction): Efficiency of the enzyme can be improved by adding more enzyme, prolonging the reaction time, or purifying DNA samples to remove inhibitory contaminants.
• Contamination with non-specific endonucleases: Non-specific endonucleases may be introduced to the DNA sample and/or the enzyme from improper handling, pipetting, etc.
•Improper reaction setup: Mix the digestion reaction thoroughly.
Find additional tips, troubleshooting help, and resources within ourRestriction Enzyme Cloning Support Center.
What are possible reasons for incomplete/failed restriction digestion?
The main reason for DNA cleavage reaction failure is the presence of contaminating inhibitors in the template DNA (for example: phenol, chloroform, detergents, ethanol, excess salts, EDTA, etc.). The best way to troubleshoot is to perform control reactions:
1) negative control (experimental DNA in the reaction buffer without the restriction enzyme) to access degradation of DNA by contaminants in the DNA template and/or reaction buffer
2) positive control reaction I (digestion of highly pure control DNA with the restriction enzyme) to access reaction conditions and enzyme activity
3) positive control reaction II (highly pure control DNA + experimental DNA + Restriction Enzyme) to access possible issues with the experimental DNA.
In addition, please check for sensitivity of the restriction enzymes to template DNA methylation.
Find additional tips, troubleshooting help, and resources within our Restriction Enzyme Cloning Support Center.
