Place the next cut size X spaces to the right of the 0/start space where X equals the size of the smallest fragments. Also write it the the position of the cut site next to the name in parenthesis. By convention cut sites are represented by a perpendicular line and labeled with the enzyme name. (Choosing the most bands isn’t required but it tends to make life easier.) Arbitrarily assign the first restriction enzyme cut site of this enzyme to the zero box at the top of the circle. Just to the left of it is the final nucleotide which is at position 0+N where N equals the total size of your plasmid.Ĥ) Begin by mapping the single enzyme digest with the most bands. The ‘box’ at the very top of the circle is given the position number 0. Take a second to imagine zooming in on this circle’s line to a focus where each point along the circumference is a box with a nucleotide base inside of it and a position number. Summing fragments across all the single enzyme digestions also lets confirm plasmid size even in the absence of a single cut site enzyme.ģ) Once you know the size of your plasmid, use a pencil to draw a circle with the total bp number next to it. (Two fragments of the same size will look like a single band on an electrophoresis gel). Digestions that sum to a different number are either the result of incomplete digestions or may have two fragments of the exact same size. Every single enzyme digest should add up to the same final plasmid size. For example, an enzyme that cuts the plasmid at two sites will produce two fragments.Ģ) Add the fragment lengths produced by each single enzyme digestion to double check your experiment and make sure that your chosen conditions allowed for complete digestions. Enzymes with more cut sites will produce multiple fragments. Such one-cut enzymes are extra useful if you’re working with an unknown plasmid as the size of the single fragment is also the size of the plasmid. In plasmids, an enzyme that has one cut site will produce a single fragment. These tell you how many cut sites (also known as cleavage sites) each enzyme has. 1) To start, focus on the single enzyme digests.
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