RE incubtation time - (Jan/24/2008 )
Hi,
According to protocols, some say that successful digestion by RE can be achieved in 1hr, but some say that it needs overnight incubation. I m quite confused. Will the later condition causes over-digestion?
I am using the MluI and XhoI from the same company. They can be added together and db digest in the same buffer.
How long should I incubate?
Thx
It depends on what you are doing. Since the enzymes you are using do not exhibit star activity (correct me if I am wrong) I would incubate over night if it is for a cloning. In my experience an efficient cleavage is extremely important for subsequent ligations. However, most of the reaction will have taken place after 1 hour if you use a proper amount of enzyme
Hi,
firstly a lot of things depend on the specific enzyme you are using. Some are fine over night, some start digesting DNA unspecifically if the incubation is too long. Here is the info i got for XhoI (NEB website):
XhoI FAQ
See the Restriction Endonucleases FAQ also.
Q1: Why isn't XhoI cutting?
Q2: Is extended digestion of XhoI recommended?
Q3: Does XhoI produce commonly used compatible ends?
Q4: How does XhoI differ from its isoschizomer, PaeR71?
Q5: What is the molecular weight of XhoI?
Q6: What is the activity of XhoI at 25°C?
Q1: Why isn't XhoI cutting?
A1: 1. Blocked by CpG methylation.
2. Supercoiled plasmids may require up to 10-fold more XhoI for complete degestoin than linear DNA.
Q2: Is extended digestion of XhoI recommended?
A2: Extended digests are not recommended due to an exonuclease or nonspecific endonuclease activity that appears to be a property of the enzyme.
Q3: Does XhoI produce commonly used compatible ends?
A3: XhoI cleaves to leave a 5´ overhang which can be ligated to fragments generated by SalI and AvaI digestion.
Info for MluI:
Q1: Are there any common problems encountered when using MluI?
A1: No.
In addition the NEB website has a lot of info on enzymes and reactions generally:
RE
And on Setting up restriction digests:
Digest set up
Read some of it, it may help you in the future...
I personally digest DNA with 3 - 5 U enzyme per µg DNA for 2 hours.
Stardust
According to protocols, some say that successful digestion by RE can be achieved in 1hr, but some say that it needs overnight incubation. I m quite confused. Will the later condition causes over-digestion?
I am using the MluI and XhoI from the same company. They can be added together and db digest in the same buffer.
How long should I incubate?
Thx
According to protocols, some say that successful digestion by RE can be achieved in 1hr, but some say that it needs overnight incubation. I m quite confused. Will the later condition causes over-digestion?
I am using the MluI and XhoI from the same company. They can be added together and db digest in the same buffer.
How long should I incubate?
Thx
I would digest them together for no longer than 1 hr. But how much DNA are you using? And also heat inactivate it as MluI can damage the ends if active.
Hi,
I've checked, below are the ingredients of my going-to-double digest reaction:
- To-be-cut DNA template: 2ug, 8.18uL
- MluI: 3uL
- XhoI: 3uL
- 10X buffer: 10uL
- h2O:75.82uL
Total = 100uL reaction
Technican of my lab said i should digest overnight, should i follow what she said?
Thx, again.
Mlu I and Xho I are perfectly friendly enzymes. Use the standard units of each enzyme per ug of DNA for 1 hr @ 37C and that will be totally fine and you'll have absolutely no problems.