How much DNA do you add to restriction digest?
In general, we recommend 5–10 units of enzyme per µg DNA, and 10–20 units for genomic DNA in a 1 hour digest.
What is the ideal concentration of DNA to be digested?
The optimal DNA concentration is between 20-100 ng/µL in the final reaction mixture. The DNA substrate should be free of contaminants or reaction components like SDS, EDTA, protein, salts, and ethanol.
How do you restriction enzyme digest?
Restriction digestion is accomplished by incubation of the target DNA molecule with restriction enzymes – enzymes that recognize and bind specific DNA sequences and cleave at specific nucleotides either within the recognition sequence or outside of the recognition sequence.
What are the steps in restriction digestion?
Restriction Enzyme Digest Protocol
- Add components to a clean tube in the order shown:
- Incubate the reaction at digestion temperature (usually 37 °C) for 1 hour.
- Stop the digestion by heat inactivation (65 °C for 15 minutes) or addition of 10 mM final concentration EDTA.
What is genomic DNA extraction?
Genomic DNA extraction methods isolate genomic DNA away from proteins, RNA and other cellular material. Such methods can involve centrifugation, vacuum or magnetic methods to separate the bound DNA from other cellular components.
How is the restriction enzyme digest protocol optimized?
Restriction Enzyme Digest Protocol Our restriction enzyme collection has been optimized for digestion using five unique buffers. When digesting DNA using a single enzyme, use the buffer supplied with the enzyme. Protocol for DNA Digestion with a Single Restriction Enzyme
How much DNA is needed for a diagnostic Digest?
A diagnostic digest typically involves ∼500 ng of DNA, while molecular cloning often requires 1 µg of DNA. The total reaction volume usually varies from 10-50 µL depending on application and is largely determined by the volume of DNA to be cut.
Which is the best way to prepare DNA for PCR?
Use an aliquot of supernatant straight from the tube (e.g., 1 ul in a 25 ul reaction) for PCR. If the results are questionable, try a 1:5 or 1:10 dilution of the DNA. Cut 1-2 mm tail and place in a 0.5 ml microfuge tube. Caution – larger pieces of tail can inhibit the PCR. Add 75 µl Alkaline Lysis Reagent.
How much DNA is cut in a restriction digestion?
The total reaction volume usually varies from 10-50 µL depending on application and is largely determined by the volume of DNA to be cut. *Pro-Tip* The amount of restriction enzyme you use for a given digestion will depend on the amount of DNA you want to cut. By definition: one unit of enzyme will cut 1 µg of DNA in a 50 µL reaction in 1 hour.