How much DNA should I 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.
Why is double digest better?
The recombinant fragments of single-digested plasmids have to be selected for their proper orientation while the double-digested plasmids ensure the proper orientation of the foreign DNA fragment. Therefore, double-digested plasmids save time in the recombinant DNA techniques, not single-digested plasmids.
What is the buffer for restriction enzyme digestion?
Buffers containing low concentrations of EDTA (1mM) are often used to protect DNA from nuclease degradation during storage, but EDTA can interfere with restriction enzyme digestion if the final concentration in the reaction is too high.
How do you know if your restriction digestion was successful?
If the digested product would be visible at a lower coordinate on the gel, it would have made things easy. You can amplify your digested fragment with primer beginning in the flankers region and with only 3-4 bp in the intern 8680 bp region. If you do not get PCR fradments, was the digestion successfully.
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.
How do you calculate digestion restrictions?
Calculate the amount of each that you need to add to a restriction digestion in order digest 5ug (5000ng) of DNA with 5 units of enzyme. For example if my DNA is at 190 ng/ul, I would need: 5000ng/190ng/ul = 26 ul of my sample.
Why we use sequential digestion during double digestion of DNA?
Since EcoRI and HindIII use different buffer, to have efficient cleavage for both of them and avoid star activity due to a change in reaction conditions, a sequential digestion is recommended. Two enzyme selected for digestion may have different buffering conditions thats why enzymes are supplied with specific buffers.
What happens if there is too much restriction enzyme?
Incomplete digestion is a frequently encountered issue when using restriction endonucleases. Incomplete digestion may occur when too much or too little enzyme is used. The presence of contaminants in the DNA sample can inhibit the enzymes, also resulting in incomplete digestion.
What is PCR and restriction digestion?
The Polymerase Chain Reaction (PCR) is commonly used to amplify a gene or DNA fragment of interest, from any source of DNA, to be cloned. These additional bases provide sufficient DNA for the restriction enzyme to bind the recognition site and cut efficiently.
What is star activity in restriction digestion?
It has been demonstrated that under extreme non-standard conditions, restriction endonucleases are capable of cleaving sequences which are similar but not identical to their defined recognition sequence. This altered or relaxed specificity has been termed “”star”” activity.
What is restriction digestion of DNA?
Restriction digestion also called restriction endonuclease is a process in which DNA is cut at specific sites, dictated by the surrounding DNA sequence. The reaction is incubated at a specific temperature required for optimal activity of the restriction enzyme and terminated by heat.