BIOB12H3 Lecture Notes - Lecture 6: Multiple Cloning Site, Ecori, Plasmid
Document Summary
Molecular biology module: restriction mapping of the unknown plasmid isolated in lab 4 using multiple restriction digests. Restriction mapping is generally used to determine whether sites for particular restriction enzymes exist and then to determine the linear order of sites. Often restriction sites comprising part of the multiple cloning site (mcs) can be used as anchor points. Consider the situation displayed in the maps above. A 5kbp bamhi/hind iii fragment is cloned into a vector. The plasmid is cut with ecori and two fragments of 2. 0kbp and 6. 0kbp are generated. Solution: since there is only one eco ri site in the vector (in the mcs) and the vector is 3kb in length, it is not possible for the 2. 0kb fragment to be generated by the vector. Instead, the ecori site must be located in the insert, 2. 0kb from the site in the vector. Use of a second anchor point and the value of double digests.
