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Frequently Asked Questions about Restriction Enzymes (4)

1. How do I choose the right buffer for double/multiple digestions with Fermentas restriction enzymes?
   
   The basic principle: if neither restriction enzyme is prone to star activity, then simply compare their activity ratings in the Five Buffer System.
    
   1. First, find the buffer from Five Fermentas Buffer System (B, G, O, R, Tango™ buffer) where both restriction enzymes show the best activity. 
      Perform a double digest simultaneously by adding a 2-fold excess* of an enzyme which rates 50-100% in the selected buffer and a 4-5 fold excess for the one which rates 20-50%:

      BglII	100% - O 50-100% - R	and	HindIII	100% - R 0-20% - O	-	R (2 u BglII, 1 u HindIII)
      PaeI	100% - B 50-100% - G	and	HindIII	100% - R 20-50% - G	-	G (2 u PaeI, 4 u HindIII)

      * - The estimated amounts refer to the restriction enzyme activity units determined on lambda DNA. Certain DNAs, such as supercoiled forms of pBR322 and pUC19, may require larger amounts of restriction enzymes for the complete cleavage.
       

   2. Or, if one is not quite sure which buffer from the Five Buffer System best suits restriction enzyme pair digestion, use the universal double digest Tango™ buffer at either a 1X or 2X final concentration. Restriction enzymes activity rates in the 1X and 2X Tango™ buffer are in the chart "Double Digestions using Universal Tango™ Buffer":

      MssI	20-50% - Tango™ 0-20% - 2X Tango™	and	HindIII	50-100% - Tango™ 50-100% - 2X Tango™	-	Tango™ (4u MssI and 2 u HindIII)
      PauI	0-20% - Tango™ 100% - 2X Tango™	and	DraI	50-100% - Tango™ 50-100% - 2X Tango™	-	2X Tango™ (1 u PauI and 2u DraI)

       

   3. If both restriction enzymes work equally well in both the 1X and 2X Tango™ buffer concentrations, then a 2X Tango™ buffer is a better choice to decrease a possibility of star activity:

      HindIII	50-100% - Tango™ 50-100% - 2X Tango™	and	VspI	100% - Tango™ 100% - 2X Tango™	-	2X Tango™ (2 u HindIII and 1 u VspI)
      PstI	50-100% - Tango™ 50-100% - 2X Tango™	and	Bsp119I	100% - Tango™ 100% - 2X Tango™	-	2X Tango™ (2u PstI and 1 u Bsp119I)

       

   4. If one restriction enzyme prefers only one concentration of Tango™ buffer, and the other shows no preferences, then choose the buffer concentration that best satisfies the requirements for both enzymes:

      Bsp120I	50-100% - Tango™ 0-20% - 2X Tango™	and	PvuI	50-100% - Tango™ 100% - 2X Tango™	-	Tango™ (2 u Bsp120I and 2 u PvuI)
      Bsp119I	100% - Tango™ 100% - 2X Tango™	and	PauI	0-20% - Tango™ 100% - 2X Tango™	-	2X Tango™ (1 u Bsp119I and 1 u PauI)

       

2. What if no single buffer satisfies the requirements for both enzymes?
   
   Then, digestions should be carried out sequentially.
   If neither a 1X nor a 2X Tango™ concentration suits both restriction enzymes, the first digestion should be performed in 1X Tango™ with the restriction enzyme that requires a buffer of a low ionic strength. When the reaction is complete add, 10X concentrated Tango™ buffer to a final 2X concentration and the second enzyme. The amount of 10X Tango™ buffer (V) which must be added in order to obtain a final 2X concentration is calculated from the formula:
       V=A x 0.125, where A = starting volume of a reaction mixture.
       SmaI (Tango™) and BglII (O) - Tango™ (1 u SmaI), incubation, addition Tango™ buffer to final 2X concentration, +BglII (1 u), incubation.
    
3. If one restriction enzyme is prone to star activity in my double digest, which buffer should I use?
   
   If there is a possibility of choosing between several buffers, then a buffer of a higher ionic strength or of a lower pH should be chosen. For several restriction enzymes in the table "Reaction Conditions for Restriction Enzymes", some buffers are marked with asterisks (*) to note that higher than a 5-fold excess of restriction enzyme should be avoided if there is no better choice:

   CfrI	100% - Tango™ 50-100% - B* 50-100% - G	and	PaeI	100% - B 50-100% - G	-	G (2 u CfrI and 2u PaeI)
   BclI	100% - G 100% - Tango™* 100% - 2X Tango™	and	EcoO109I	100% - Tango™ 100% - 2X Tango™	-	2X Tango™ (1 u BclI and 1 u EcoO109I)

    

4. How should I choose a buffer for double digestion for restriction enzymes supplied with their individual optimal buffers?
   
   For most Fermentas restriction enzymes supplied with individual optimal buffers either a buffer from the Five Buffer System or 2X Tango™ buffer can be chosen to perform the double digestion reaction (See table "Double Digestions using Universal Tango™ Buffer").
       Ecl136II and Eam1105I - Tango™ (2u Ecl136II and 2 u Eam1105I)
       BamHI and HindIII - 2X Tango™ (2 u BamHI and 2u HindIII)
       Cfr9I and SmaI - Tango™ (4 u Cfr9I and 1u SmaI)
       BamHI and EcoRI - 2X Tango™ (2 u BamHI and 1u EcoRI) or O (4 u BamHI and 1 u EcoRI)
    
   Only for BseXI, PagI, ScaI and SduI we recommend digestions in their individual optimal buffers to avoid appearance of star activity.
    
5. What is the highest enzyme excess that can be used in the digestion reaction at no risk of star activity?
   
   Avoid higher excess (> 5-10 fold) of restriction enzyme when working in a lower ionic strength buffer (B or G) or a higher pH buffer compared to its optimal.
   The specific info for each restriction enzyme is given in its catalog description, on certificates of analysis and in a summary table "Reaction Conditions for Restriction Enzymes".

Updated gegužės 09, 2007 16:41