What is the purpose of double digestion?
Digesting a DNA substrate with two restriction endonucleases simultaneously (double digestion) is a common timesaving procedure. Selecting the best NEBuffer to provide reaction conditions that optimize enzyme activity as well as avoid star activity associated with some enzymes is an important consideration.
Can you use two restriction enzymes at once?
To be able to clone a DNA insert into a cloning or expression vector, both have to be treated with two restriction enzymes that create compatible ends. If one of the enzymes is a poor cutter or if the sites are separated 10 base pairs or less, the digestions should be performed sequentially.
How is a sequential digest performed?
Setting up a Sequential Digestion Set up a reaction using the restriction endonuclease that has the lowest salt concentration in its recommended buffer and incubate to completion. Add the second enzyme and incubate to complete the second reaction.
How do you test for double digestion?
Digestion: 1ug of DNA+2uL of Cut smart buffer (10x if your using NEB product)+1uL of each enzyme (according to your mentioned stock conc.)+ rest make up with sterile milliQ. Incubate at 37oC for 3 hrs, estimate the digested products concentration. Verify that both enzymes using the same reaction buffer.
Why do you use 2 restriction enzymes?
The use of 2 different enzymes makes self ligation of the vector impossible and makes the insertion unidirectional. Whereas in the case of single digest, selfligation occurs and insertion may occur in both ways.
What 3 reagents are necessary to perform a restriction digest?
Reagents
- Liquid DNA aliquot of your plasmid of interest (see below for recommend amounts)
- Appropriate restriction enzyme (see manufacturer’s instructions for proper ammount)
- Approrpriate restriction digest buffer (see manufacturer’s instructions)
- Gel loading dye.
- Electrophoresis buffer.
- Pipet tips.
What is double restriction digestion?
Double Digests. Digesting a DNA substrate with two restriction endonucleases simultaneously (double digestion) is a common timesaving procedure. Selecting the best NEBuffer to provide reaction conditions that optimize enzyme activity as well as avoid star activity associated with some enzymes is an important consideration.
What is a Double Digest?
A double digest means that you cut your plasmid with two different restriction enzymes at the same time. As you already have a putative map of the plasmid, you can predict which would be the expected bands.
What is enzyme digestion protocol?
Protocol for DNA Digestion with a single restriction enzyme 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 10mM final concentration EDTA .