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Frequently Asked Questions about Restriction Enzymes (3)

1. What is the Fermentas Five Buffer System?

   It is a simple color coded restriction enzyme digestion Buffer System composed of B (blue), G (green), O (orange), R (red) and Tango™ (yellow) buffers and is an optimal system for dependable and reproducible Fermentas enzyme performance.
    
   Every Fermentas restriction enzyme performs to 100% of its certified activity in its recommended buffer, as well as to 20-100% in the universal double digest Tango™  buffer. Although 18 Fermentas restriction enzymes require unique buffers for optimal performance, they perform no less than 50% in one of the Five System Buffers.
    
   For your convenience, each Fermentas restriction enzyme is supplied with 2 buffers: its recommended buffer and the universal double digest Tango™ buffer.
    

2. What are the advantages of having BSA pre-added to the restriction enzyme buffers?

   
   BSA enhances the stability and performance of many restriction enzymes, binds impurities that are sometimes present in DNA preparations (especially in relatively crude DNA preps). Fermentas pre-adds BSA to restriction enzyme buffers for your convenience to enhance complete digestions thus, no prolonged incubations or large enzyme excesses are required.
    

3. Each Fermentas enzyme is supplied with two 10X buffers: the recommended and universal double digest Tango™. Why?
   
   To enhance the convenience of working with Fermentas Restriction Enzymes (and later Modifying Enzymes) every Fermentas restriction enzyme is packaged with both:
   1. The recommended buffer - one of the Five Buffer System buffers or an individual optimized buffer (for AarI, BamHI, BfuI, Bpu10I, BseXI, Bsp143I, Cfr9I, Cfr10I, Eam1105I, Ecl136II, Eco52I, EcoRI, KpnI, SacI, ScaI, SdaI, SduI and TaqI).
       
   2. Tango™ buffer, the universal double digest buffer. This is a unique buffer which is:
      • an optimal buffer for multiple digestions. 98% of Fermentas restriction enzymes perform 20-100% in a 1X or 2X Tango™ buffer, including those that are supplied with an individual optimal buffer;
      • compatible with Fermentas DNA/RNA Modifying Enzymes. Most Fermentas Modifying Enzymes perform 100% in Tango™ buffer.
         
4. What is the influence of buffer additives on restriction enzyme performance?

   1. Triton X-100:
      It is included into buffers for Bsp143I and KpnI.
      Note that though TX-100 has no influence on the activity of most restriction enzymes, but it inhibits the activity of BspTI, PvuII and XbaI about 2-3 fold.
       

   2. DTT:
      It should be included into the buffer of Esp3I, but has no influence on the activity of most restriction enzymes.
      Note DTT is not supplied with the enzyme since it is not stable in solution. Furthermore, decomposition products of DTT may inhibit the activity of some restriction enzymes. In order to avoid such undesirable effects, a freshly prepared DTT solution should be added directly to the reaction mixture before digestion.
       
   3. SAM:
      It stimulates the activity of BplI, BseMII, Eco57I. SAM is supplied along with the recommended buffer.

Updated lapkričio 29, 2006 13:06