Restriction endonucleases such as EcoRI recognize specific palindromic sequences and cleave a phosphodiester bond on each strand at that sequence. After digestion with a restriction endonuclease the resulting DNA fragments can be separated by agarose gel electrophoresis and their size can be estimated. A restriction map is generated by using the fragment size data to determine the location of the specific endonuclease recognition sequences on the plasmid.
Each restriction enzyme requires specific reaction conditions for optimum activity. One of the most important reaction conditions which varies between different restriction enzymes is the salt (usually NaCl) concentration. Enzyme buffers are specifically formulated to provide the salt concentration for optimal enzyme activity. It is important, therefore, that the correct buffer solution is used for a particular restriction enzyme.
Listed below is a general procedure for conducting restriction digests. Keep in mind, however, that one should always consult the manufacturers recommendations of optimal conditions for each restriction enzyme (See Resources for more information on restriction enzymes).
1. For each digest combine the following solutions in a microcentrifuge tube.
|deionized water||6 uL|
|10x reaction buffer||1 uL|
|Plasmid miniprep DNA||3 uL|
3. Tap the tube to mix the contents and then pulse down the tube in a microcentrifuge.
4. Incubate at 37 deg C for 2 h to overnight. After incubation place
tubes in the freezer.
The First Digest of the Semester
Your first restriction digest will be a double digest to determine the size of your insert (the cDNA). A double digest is one where two restriction enzymes are used to digest DNA in a single reaction. In this case you will be using EcoR I and BamH I. There is only one site in the plasmid vector for each of these enzymes and they are located on either side of your insert DNA.
Digesting with both will cut the insert from the vector. Next week you will determine the size of the insert by seperating the digested DNA on an agarose gel. The insert may also contain a site for one or both of these enzymes and if so, the insert will be cut into multiple pieces. By adding up the sizes of each fragment you can still determine the size of the insert.
1. In a microcentrifuge tube place 6 uL of deionized water, 1 uL of 10x EcoR I reaction buffer , 3 uL of your plasmid miniprep, 0.5 uL of EcoR I enzyme and 0.5 uL of BamH I enzyme. The enzymes and buffer are stored in the freezer. The tube with the buffer has a black top. The tube with EcoR I enzyme has an "E" with a black dot in top and the tube with the BamH I enzyme has a "B" and a brown dot on the top.
2. Tap the tube to mix the contents and then pulse down the tube in a microcentrifuge.
3. Incubate in the 37 degree incubator for 1 hour. Either store it in the freezer or prepare it for agarose gel electrophoresis.