Enzyme units - (Jan/19/2006 )
I need some help regarding restriction enzymes and units! I do understand the definition but it's kind of hard to apply to a reaction! For ex., if you ahve an enzyme with 10U/microliter, how much should you add in a 10microliter digestion reaction!? Let's say that i am using 1 microliter of 100ng/microliter palsmid.
Thanks guys:)
it means that with 1 uL you could digest 100 samples of 100ng/uL.
if you have only one sample, use 1 uL
if you have several samples, you can prepare a mix where you put only 1 uL of enzyme and distribute this mix on all your samples.
you will not put too much enzyme (too much glycerol however is not good. 1 uL in 20 uL is fine, 1 uL in 10 uL : I don't know. let's see what others will tell.
However you can save money if you prepare a mix instead to put 1 uL in each tube.
if you have only one sample, use 1 uL
if you have several samples, you can prepare a mix where you put only 1 uL of enzyme and distribute this mix on all your samples.
you will not put too much enzyme (too much glycerol however is not good. 1 uL in 20 uL is fine, 1 uL in 10 uL : I don't know. let's see what others will tell.
However you can save money if you prepare a mix instead to put 1 uL in each tube.
I agree with all of the above. But I remember I was taught that the limit was that Restriction enzyme should not make more than 10% of the final mix. It always worked like this for me, but I always try to stay as far from the limit as possible. But sometimes you want a complete digestion and fast (could one put enough enzyme to have the digestion ready yesterday?)
the way the units are expressed doesn't help too much in most cases, as usually it's given as amuont of restriction enzyme, that will cut a certain amount of lambda dna in a certain time.
if you want to compare, you have to check how many sites there are in lambda for your enzyme and how many aites are on your dna and than start calculating 
I don't understand why you want to digest the plasmid in only 10 ul final volume?
I always do in 30 or 50 ul .
and use 1 unit of enzyme per reaction (0.1ul of enzyme in case the stock is 10U/ul).
because I always have several samples, I prepare a mix therefore I don't need to pipet very small volumes of enzymes.
1ul per reactio per 10ul final voulme is indeed to much.
You are at the limit of maximum enzyme possible, 10% of total volume. more than this means that you have too much glycerol in the reaction which inhibits digestion.
I always do in 30 or 50 ul .
and use 1 unit of enzyme per reaction (0.1ul of enzyme in case the stock is 10U/ul).
because I always have several samples, I prepare a mix therefore I don't need to pipet very small volumes of enzymes.
1ul per reactio per 10ul final voulme is indeed to much.
You are at the limit of maximum enzyme possible, 10% of total volume. more than this means that you have too much glycerol in the reaction which inhibits digestion.
Yes, that's what I thought, that 0.1 microliter per 10microliter reaction should be enough, thanks! I think I will do my reactions in 30 ml in the future. These 10 microliter reactions seem not to work good for me, i get only partially digests:(, but that can be for various reasons.
Thanks for the comments!