Restriction digest calculations?

Question

I have to do a restriction digest of 0.2 μg/μL DNA with EcoR I (2.5 U/μL) and Hind III (10 U/μL). How can I calculate the exact amount ( in μL) of the DNA, EcoR I, Hind III, 10xbuffer and water I have to use? By definition, the DNA should be 0.5- 2.0 μg and the final volume 20-40 μL.
Thanks in advance!

P.S: What if the enzyme needs BSA? How does this interfere with the calculations?

Answer 1

The definition of a unit (U) of the EcoRI restriction enzyme is the amount that will digest 1 microgram of lambda DNA in an hour at 37C in a 50 ul volume. In practice, generally you use an excess (5-10 fold) of the restriction enzyme to overcome variations in DNA purity and content, so that you get as complete digestion as possible. Typically the volume is 20 ul. So, if you want to digest 1 ug of DNA, then use 10 units of enzyme in a 20 ul volume. So, for 0.2 ug/ul of DNA, take 5 ul (0.2 ug/ul x 5 ul = 1 ug)) add, 4 ul of EcoRI at 2.5 U/ul (2.5 U/ul x 4 ul = 10 U), add 2 ul of 10x buffer, and 9 ul of nuclease-free water to a final volume of 20 ul and incubate for an hour.



Similar reasoning for HindIII, except use 1 ul of HindIII and 12 ul of nuclease-free water with 2 ul of 10x buffer and 5 ul of DNA for a total volume of 20 ul.



If you need to add BSA, then adjust the volume of water to compensate.



Here are some webpages that may help:



http://labtutorials.org/2009/03/15/restriction-enzymes/



https://www.neb.com/protocols/2012/12/07/optimizing-restriction-endonuclease-reactions

Answer 2

Fermentas Double Digest

Answer 3

What EcoRI do you have that's only 2.5 units/uL? Most are 20. Maybe fermentas fastdigest is only 2.5



Anyway, there's no problem putting a little excess enzyme in. Are you planning to do a double digest? If so you need to make sure they can use the same buffer. Check the website of the enzyme manufacturer for that. If the enzymes come from different companies, don't try double digest.



Generally I do a 50ul reaction:

1ul of enzyme (1 of each if it's a double digest; but should not exceed 2ul)

5ul of 10X buffer

DNA (don't exceed the number of ug that your limiting enzyme has units/ul)

(so in your case for EcoRI, maximum of 2.5ug, which is 12.5ul at 0.2ug/uL)

(ok to use less than the maximum if your enzymes are very robust and/or your DNA conc low)

(max volume of DNA you can use is the amount that would bring the total volume up to 50ul)

BSA (check to see from the manufacturer whether it's required--and what amount to use--usually 0.5ul)

water to total of 50ul (50ul minus all the other stuff you put in... if you use a lot of DNA, sometimes this is 0)



also... keep in mind that if you're trying to cut a linear piece of DNA near the end, you can't go all the way to the units/uL capacity of the enzyme (2.5ug DNA for your EcoRI), because that number is assuming your cutting a circular plasmid, which cuts much easier. If you are, I wouldn't exceed the capacity of 1/4 of the total units/ul of the enzyme--in your case 0.625ug



for your 10 units/uL enzyme, you CAN lower the amount of enzyme so it fits exactly the amount of DNA you put in (or cram 10ug of DNA into the reaction.... way more than you need).... but don't bother. As I said it's fine to have excess enzyme. 1ul / 50ul reaction works well, just don't exceed the proper amount of DNA that goes in.