Redasoft Corporation

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Whether you're using restriction enzymes for cloning, SNP analysis, RFLP, or restriction mapping, choosing the right ones is an important first step. Here are some tips to help ensure your restriction digest works optimally, the first time around. 1. Identify restriction sites in your insert and vector, for cloning experiments. See if compatible sites are available and what size bands can be expected. Match the best restriction sites to enzymes you have in the lab. 2. Aim for restriction enzymes that cut with sticky ends for cloning a fragment into a vector. If no compatible sticky ends can be found, blunt-cutting enzymes can be used but ligation will be less efficient. 3. Plan your diagnostic digests - which enzymes you will use to verify that the cloning experiment was successful. Make sure you will be able to tell if it was inserted in the correct location and orientation. 4. For cloning, check for enzyme uniqueness in both the backbone and insert DNA to avoid cuts you don't want. 5. To insert a fragment into a vector, use enzymes with different sticky ends. This will make sure your DNA ends up in the vector in the correct orientation, and will prevent the backbone from closing on itself. 6. For mapping genomic DNA, YACs, BACs, or P1s, use restriction enzymes with 6 bp recognition sequences. These will cut on average every 4096 bases (vs. every 256 bases for a 4 bp recognition sequence enzyme), which is a suitable size range for cloning. 7. If you are using more than one restriction enzyme in one reaction, make sure they are both at least 75% active in the same reaction buffer and at the same reaction temperature. If not, run one digestion, purify the reaction products, then run the second digestion. 8. When you've chosen the recognition sequence you want to cut, check all the isochizomers (restriction enzymes that recognize the same sequence) to find one that works best with your desired reaction conditions. 9. Check for methylation sites in your DNA sequence, as methylation can inhibit restriction enzymes that are sensitive to it. The dam methyltransferase sequence is GA*TC, and the dcm methyltransferase sequences are CC*AGG, CC*TGG. Check your enzyme supplier for information on dam/dcm sensitivity for each enzyme. 10. If the reaction is not working, try some control reactions: experimental DNA with no restriction enzyme (to check for DNA degradation) and control DNA with enzyme (to check for enzyme activity, where the control DNA has many cut sites for the enzyme). These tips should give you a good head start. Redasoft Visual Cloning is another useful tool, integrating your DNA sequences with a virtual cloning tool and restriction enzyme selection process. Visual Cloning makes it easy to simulate your restriction enzyme experiment in just a few clicks. Download a free trial here ( http://www.redasoft.com ). References http://www1.qiagen.com/literature/qiagennews/0301/1017317_QN301_ITJPDIS-24-27.pdf http://www.neb.com/nebecomm/tech_reference/restriction_enzymes/setting_up_reaction.asp http://openwetware.org/wiki/Cloning_Checklist http://oardc.osu.edu/sallymiller/Extension/presentations/Ins%20and%20Outs%20of%20RE.pdf http://bitesizebio.

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