Restriction Digestion of DNA
互联网
Restriction enzymes are expensive ($40 to $200 a vial): keep the enzymes at -20 C. Plan your digests to be small and convenient. The digest is composed of DNA sample (volume should not be more than about 1/3 of the total digest volume if possible). 1/10 digest volume of 10X restriction buffer, the appropriate number of units of enzyme (the enzyme volume should not exceed 1/10 of the digest volume), and sufficient H2O to bring the mix up to the digest volume.
Be sure to get all other component of your digests into the MFT before getting the enzyme. Then get the appropriate Stratacooler out of the freezer, and remove the necessary amount of enzyme with a new pipet tip while trying to leave the vial in the Stratacooler well as much as possible.
The minimum amount of enzyme necessary should be used. The one unit of enzyme activity is defined as the amount needed to digest 1 µg of a specific DNA in a 50 µl digest at the appropriate temperature (usually 37 C). Also the more concentrated the enzyme (i.e. the smaller the digest reaction volume) the higher rate of digestion. DNA is DNA: there is only an activity difference if 1) the DNA used to define the units differs greatly in base ratio from the sample DNA, and 2) the enzyme's target site is GC rich or poor so that the substrate (the base recognition sequence) concentration is higher or lower (the sample DNA will then digest faster or slower). Methylation and the purity of the DNA can also effect the enzyme activity. The purer the DNA the less enzyme will be needed: BRL showed that 3 sequential NH4Ac:EtOH precipitations reduced the amount of enzyme needed 10 fold over no precipitation.
A 4 fold over digest won't be too expensive: i.e. 2 times the necessary enzyme and at least 2 hrs. So, use 1 or 2 units per µg DNA for 2 or more hr; it is better to use 1 unit per µg DNA and digest overnight. For large genomic digests, use 0.5 unit per µg DNA and digest overnight. (However, use 2.5 units per µg cotton genomic DNA and digest overnight. It seems that digestion of cotton genomic DNA requires more than standard amount). Standard digestion temperature is 37 C (but see restriction enzyme list for specific information).
If multiple digests are to be done with the same enzyme, pool the 10X restriction buffer with a portion of the H2O, add the total amount of enzyme required to this mix and pipet this into the prepared sample containing the DNA and the balance of the H2O.
Stop the digest with 0.1 volume of tracking dye, if desired, add 0.2 volume of 0.5 M Na3EDTA, for running a gel
