Units of my RE -10,000 units. How much RE is required to digest 10ul of DNA?
Hi!
Quantity of DNA always expressed in micro grams not in micro liters.
Amount of RE needed is proportional to the quantity of DNA taken. Sometime back one person asked question related to same topic.
As per the manufacturer the following is definition. Take that many units or slightly more, 1 or 2 units extra for complete digestion. Refer Sambook et. al. A good book for mol. bio. techniques. You get A-Z information.
Unit Definition
One unit is defined as the amount of enzyme required to digest 1 µg of λ DNA in 1 hour at 37°C (Temp. varies as per the enzyme taken) in a total reaction volume of 50 µl.
This also depends on the DNA concentration you have. In general, 0.5 µl of an RE is enough for a miniprep digestion. You should also keep in mind not to use more RE than 10% of the final reaction volume, because of the glycerol within the storage buffer.
But these are just general recommendations. You could also test several RE volumina to check the best result.
Hi!
Quantity of DNA always expressed in micro grams not in micro liters.
Amount of RE needed is proportional to the quantity of DNA taken. Sometime back one person asked question related to same topic.
As per the manufacturer the following is definition. Take that many units or slightly more, 1 or 2 units extra for complete digestion. Refer Sambook et. al. A good book for mol. bio. techniques. You get A-Z information.
Unit Definition
One unit is defined as the amount of enzyme required to digest 1 µg of λ DNA in 1 hour at 37°C (Temp. varies as per the enzyme taken) in a total reaction volume of 50 µl.
1 ul of RE will be sufficient for the digestion. recipe for this:-
10 ul of DNA
7 ul Nuclease free water
2ul buffer provided with RE
1 ul RE
We have tried lot of time to restrict the DNA. This recipe gave good results.
You need to provide some more info. In standard notation one unit of RE cuts one microgram of DNA at 37 degree in an hour or so. In your case if you know the concentration of your DNA then it would be easier to restrict DNA in quality terms. You can use NanoDrop or NanoVue and if you don't have any such instruments just run 1 or 2 uL of your DNA on agarose. It will give you an idea of concentration of DNA. Then accordingly set up the restriction reaction. Believe me it saves a lot time than doing it blindly.
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i got the point.What is the ideal volume of every component of reaction mixture?
Good Day,
I recommend these volumes for 15 uL of DNA (already tested in my work) so you can divide per 1.5 each of these volumes to adjust them for a voume of 10 uL DNA:
Extra Pure water 19 uL, Buffer (Provided with RE) 4uL, PCR prodcuts (or DNA sequence) 15 uL, RE 2 uL.
Sincerely Yours
You need need to specify the quantity of DNA in ug and not in volumes for restriction digestion . The rule of thumb is that 1U is enough for about 1 ug of DNA in a 50 ul vol. Here the main point is that the glycerol percentage in the reaction vol should not exceed 5.
Just Measure your DNA concentration and Try to understand the definition of unit/U of restriction enzyme to be clear.
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