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Frequently Asked Questions about Restriction Enzymes (7)

1. What should I know to set up a successful restriction reaction:
   
   Hints on DNA preparation purity, restriction enzymes, buffers, reaction volume, mixing, incubation temperature and time, stopping a reaction and much more.

• About DNA
  Two things should be considered:
  • DNA preparations to be digested should not contain contaminating substances such as organic solvents, excessive salts or EDTA that can inhibit restriction enzyme activity.
  • DNA methylation. For more information see Restriction Enzymes: Troubleshooting.
     
• About enzyme
  All restriction enzymes should be stored at -20°C or on ice when not in freezer. A restriction enzyme should always be the last to be added to the digestion reaction mixture. The amount of enzyme to be added to achieve the complete cleavage depends on the nature and purity of DNA substrate. For more info see Restriction Enzymes: Troubleshooting.
   
• About reaction buffer
  A color coded 10X optimal buffer and a 10X universal double digest Tango™ buffer is supplied with each Fermentas restriction enzyme. A recommended buffer is always used at a 1X final concentration, while the universal double digest Tango™ buffer can be used either at a 1X or 2X final concentration (depending on restriction enzymes in a digest). All buffers from the Fermentas Five Buffer System have BSA already pre-included to buffers. For more info see Five Buffer System.
   
• About reaction volume
  For the right choice of a reaction volume, which usually depends on the needs of a particular experiment, the following is important:
  • working with small volumes, excessive amounts of enzyme and/or glycerol should be avoided, see Restriction Enzymes: Troubleshooting, Star Activity.
  • viscous DNA solutions inhibit enzyme diffusion and can reduce enzyme activity.
  • too low DNA concentrations that can fall below the Km of the restriction enzyme and also affect enzyme activity.
    Reaction volumes of 10-50 µl per microgram of DNA are the most commonly used.
     
• About mixing
  In order to achieve the uniform reaction conditions a restriction reaction mixture should be properly mixed up. Gentle pipetting of a reaction mixture for several times followed by quick spin down in microcentrifuge is preferential to its vortexing.
   
• About incubation temperature
  The recommended incubation temperature for the majority of restriction enzymes is 37°C, unless otherwise indicated. Restriction enzymes isolated from psychrophilic or thermophilic microorganisms require optimal reaction temperatures that are higher or lower than 37°C. For incubations carried out at high temperatures for longer than 1 hour, it is recommended to cover the reaction mixtures with a drop of mineral oil to prevent evaporation. For enzymes that have optimal temperatures other than 37°C, Fermentas provides information on activity rates of these restriction enzymes at 37°C in the table " Activity of Mesophilic and Thermophilic Enzymes at 37°C". This information is also provided in the catalog description of each enzyme and on the Certificate of Analysis packaged with each enzyme. This type of information is particularly useful when performing double digests.
   
• About incubation time
  Usually restriction enzyme digestions are incubated for at least one hour. Complete digestions can be achieved either with less enzyme units prolonging incubation time or with an excess of enzyme shortening incubation time.
  For more info see "Stability during Prolonged Incubation".
   
• About stopping a reaction
  If the DNA digested shouldn't be used in further reactions, then no further manipulations of the digested DNA are to be performed, then the reaction can be stopped by adding 6X Loading Dye Solution, Cat #R0611 (10 mM Tris-HCl (pH 7.6), 0.03% bromophenol blue, 0.03% xylene cyanol FF, 60 mM EDTA, 60% glycerol) to a 1X final concentration.
   
  If further reactions on the digested DNA should be performed, then it is necessary to inactivate the restriction enzymes. As a rule, thermal inactivation (at 65°C or 80°C for 20 min) of the reaction mixture with restriction enzyme is the most convenient method. For inactivation of the following thermostable enzymes: AdeI, BcuI, BglII, BseLI, Cfr10I, TaaI, TaiI, TasI, TaqI, TauI and Tru1I, we recommend phenol/chloroform deproteinization and ethanol precipitation. For more information on thermostable restriction enzymes see a section "Thermal Inactivation".
   

2. Are Fermentas restriction enzymes supplied at a standardized concentration?
   
   The majority of Fermentas produced restriction enzymes are supplied at a standardized 10 units/µl, some - at a 1-5 units/µl.
   Many restriction enzymes are available at a HC format (50 units/µl), which is helpful for compensating for lower activity rates of thermostable enzymes digestion at non optimal temperatures, as well as for digesting large amounts of DNA.
   See the table "Activity of Mesophilic and Thermophilic Enzymes at 37°C".

Updated gegužės 09, 2007 13:03