I think you won't need that much of enzyme.

But some information is missung. What amount of DNA will be the restriction reaction? Does the used enzyme have star activity?

Also I would guess that by overloading the gel with digested DNA you are going to loose a large part of your product in the smear.

Usually (if I am right) you won't get high concentration of purified DNA by Gel extraction. Depends of of course on your definition of "high concentrated"

Hi Soundarya

I agree with Normann, you can scale up your reaction if you consider the right enzyme/DNA ratio, and you can also increase the time of digestion. But generally the issue is the purification AFTER the digestion, as Normann wrote. Digested product yield can be affected by the purification kit (e.g. spin columns-based). What kind of DNA are you dealing with? Plasmids can be also purified with other kits (just run a small aliquote on gels to check if the digestion worked, and use the other part for purification without "moving" to the gel).

I strongly suggest you scale up the reaction. As digestion that contain high amounts of glycerol will cause star activity (the source of the glycerol is the enzyme storage buffer which contains 50% glycerol, you should try not to exceed 5% v/v glycerol concentration).

The concentration of Plasmid DNA i need to digest is 50ug. If I digest 10ug of plasmid I loose some amount in the digested fragment and elution from the gel yields me nothing. But if I increase the reaction volume and should i run multiple gels and elute them all together??

Soundarya Priya,

Previously, I had probelms with the concentration of my digested DNA parts (Plasmid DNA). I optimized my concentrations by adding less or no water to my digestion reaction and it worked pretty well, as far as the units of enzyme you are using and the concentration (ng) of your DNA (to be digested) are according to the recommended protocol of the enzyme, I think your digest should work.

Also, by increasing your reaction voulme by increasing your enzyme units could exhibit star activity. Usually, in our lab we increase enzyme volume upto two times only if the enzyme efficiency is low. Which reminds me to first optimize your enzyme digestion activity using a control DNA (you must have a positive control somewhere, eg a circular plasmid containing a site for your enzyme), since enzymes should be optimized every 6-8 months of storage for good lab pracitices.

I can suggest all this by my very own experience after infite times of failures. Some might not agree which is okay ^_^

Good luck!

Its not at all clear why you need so much plasmid and its purpose. So, for example, if you are preparing it for transfection, you probably dont need to do anything with it, apart from heat inactivating the restriction endonuclease. For that amount of plasmid, ethanol precipitation will remove buffers that might interfere with transfection, if you have to. But of course maybe you are doing something else, and then the advice would be different.

You can always increase the concentration of your desired digested product using a speed-vac or putting the tubes in a warm heat block with the lid open. Out of curiosity, why does the concentration need to be so high?

the answer to your question is - no you can not,

enzymes are in 50% glycerol and will NOT work in concentration higher than 5%.

try some HF enzymes which have more units per ul

or scale up the reaction for 10ug i would use 100ul total volume at least with 10ul buffer and 10ul enzyme...

50ug is insane......

Usually the enzymes need to be less than 10% of the overall reaction volume. But you typically don't need very large volumes of enzyme. It's a good idea to check the activity/unit of the enzyme as mentioned by the manufacturer's. Also, you don't need to gel elute, as there may be a lot of product lost, you can simply column purify. But if you want to use the digested DNA for ligation, inactivating the enzyme prior to ligation is also sufficient to achieve a good ligation. I have digested upto 5.5 ug of DNA with 1U of FD restriction enzymes. To check for cleavage loading a small aliquot of the digestion mix on the gel and checking for any remaining supercoiled plasmid should work well instead of loading the entire 50ug of digested DNA.

For you to have a high amount of restriction product, you need to start with a high amount of template in the restriction reaction. Work on increasing the template concentration!

In addition, I think it is not advisable to just increase the enzyme concentration in the hope that you will get higher restriction product. Enzymes have a specific activity depicting the amount of enzyme that can digest a given amount of template in a specific amount of time. Using alot of restriction enzyme may be wasteful for enzymes which are normally expensive.

I suggest that you increase your template and use the correct of enzyme for the concentration of the template and reaction volume. After the restriction, you can concentrate the reaction by removing excess water using a vacuum extractor. In that way, you can have a high amount of your restriction product.

you need to make a good balance between the DNA concentration and the enzyme activity or unit. Checking the unit of the enzyme form the manufacturer is a good idea. In principle, increasing the concentration of the enzyme should accelerate the reaction rate, but for digesting a template DNA, you don't need high enzyme concentrations, just make sure that the used DND concentration is suitable for your enzyme activity.

I think has others have said, it is probably best to scale up. There is the issue with glycerol concentration but also the issue with low level contaminants in your DNA. If you need to concentrate your DNA after the digestion, you can do an ethanol precipitation. I would do at least 200ul reaction if not more for 50ug of DNA. However unless you are loading a gel with really large lanes, you can't load that much DNA on a normal gel. For a normal analytical gel about 1ug of DNA is the maximum. 50ug is a LOT of DNA.

Yes you can, but make sure that final concentration of glycerol does not increase more than 5 % V/V, which comes from your enzyme stock.