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question about restriction enzyme digest


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#1 newuser1234

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Posted 18 November 2009 - 03:36 PM

I know there are many definitions of a unit of enzyme but the definition i was given in class is "the amount of enzyme needed to digest 1ug of DNA in 1 hour at optimal temperature in a 50 uL reaction volume."

We were given a question on an exam where we were to calculate the amount of enzyme needed for a 20 uL digest. I took into account the reduction of volume and had points taken off because my professor said that the new volume did not have to be taken into account. My question is when does the final volume of the digest become a factor in how many units of enzyme you add? Using my professor's answer, I could perform that digestion in a bath tub as long as I keep the amount of enzyme and DNA the same which cannot be true. Any help would be greatly appreciated! :P

#2 jaya2020

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Posted 30 May 2010 - 12:11 AM

I know there are many definitions of a unit of enzyme but the definition i was given in class is "the amount of enzyme needed to digest 1ug of DNA in 1 hour at optimal temperature in a 50 uL reaction volume."

We were given a question on an exam where we were to calculate the amount of enzyme needed for a 20 uL digest. I took into account the reduction of volume and had points taken off because my professor said that the new volume did not have to be taken into account. My question is when does the final volume of the digest become a factor in how many units of enzyme you add? Using my professor's answer, I could perform that digestion in a bath tub as long as I keep the amount of enzyme and DNA the same which cannot be true. Any help would be greatly appreciated! :lol:


hi

It is the amount of DNA that is taken into account while performing a digest and not the final volume of the reaction mixture. Generally it is taken as 50 uL, but in actual practice the amount is generally 10 -15 uL. If the volume of the reaction is minimised, it dosent mean the conc of the DNA is minimised or if the volume is increased there is the dilution of the DNA. The enzyme depends on the concentration of the DNA and not on the volume mixture. Hope this would have answered ur question.

Rgards,

jaya




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