Ligation restriction enzymes
Pharmaceutical Science Year 2
Date: 7th- 14th of February 2014.
Title: Ligation of Lambda DNA pre-digested with EcoRI and HinDIII. Restriction of Lambda DNA with restriction enzymes.
Aim: The objectives of this experiment are:
Become more familiar with using micropipettes.
Use restriction enzymes to cut DNA at specific sites.
Use Ligase to rejoin some of the cut/separated DNA fragments.
Learn to separate DNA using electrophoresis.
Introduction:
Restriction enzymes are proteins which cut dsDNA at specific regions depending on the enzyme used, determined by the nucleotide sequence of the DNA, i.e. each enzyme recognises specific nucleic acid sequence of 4,6,8 nucleotides.
Restriction enzymes are taken from cells. Because restriction enzymes are cut within a molecule they are often known as restriction endonucleasses.
Restriction enzymes are found in a variety of different strains of bacteria. There biological role is to perform cell defence, this is where they function to destroy any foreign DNA in bacterial cells. Restriction enzymes ‘restrict’ foreign DNA that enters a cell.
Restriction enzymes are used today in biotechnology to cut DNA into smaller strands to study fragment length differences. This is known as restriction fragment length differences (RFLD), or also known as gene cloning.
There are three types of restriction enzymes:
Type one cut the DNA at random regions as far as one thousand or more base-pairs from the recognition site.
Type two enzymes are often used in biotechnology; they cut DNA within a recognised sequence without any need for energy (ATP). This type is a very simple and small enzyme.
Type three cuts DNA at approximately twenty five base-pairs from the recognition site.
Type one and type two restriction enzymes require ATP and are quite large enzymes that have multiple subunits.
Restriction enzymes are named based on the bacterial species that they are isolated from. For example,
Date: 7th- 14th of February 2014.
Title: Ligation of Lambda DNA pre-digested with EcoRI and HinDIII. Restriction of Lambda DNA with restriction enzymes.
Aim: The objectives of this experiment are:
Become more familiar with using micropipettes.
Use restriction enzymes to cut DNA at specific sites.
Use Ligase to rejoin some of the cut/separated DNA fragments.
Learn to separate DNA using electrophoresis.
Introduction:
Restriction enzymes are proteins which cut dsDNA at specific regions depending on the enzyme used, determined by the nucleotide sequence of the DNA, i.e. each enzyme recognises specific nucleic acid sequence of 4,6,8 nucleotides.
Restriction enzymes are taken from cells. Because restriction enzymes are cut within a molecule they are often known as restriction endonucleasses.
Restriction enzymes are found in a variety of different strains of bacteria. There biological role is to perform cell defence, this is where they function to destroy any foreign DNA in bacterial cells. Restriction enzymes ‘restrict’ foreign DNA that enters a cell.
Restriction enzymes are used today in biotechnology to cut DNA into smaller strands to study fragment length differences. This is known as restriction fragment length differences (RFLD), or also known as gene cloning.
There are three types of restriction enzymes:
Type one cut the DNA at random regions as far as one thousand or more base-pairs from the recognition site.
Type two enzymes are often used in biotechnology; they cut DNA within a recognised sequence without any need for energy (ATP). This type is a very simple and small enzyme.
Type three cuts DNA at approximately twenty five base-pairs from the recognition site.
Type one and type two restriction enzymes require ATP and are quite large enzymes that have multiple subunits.
Restriction enzymes are named based on the bacterial species that they are isolated from. For example,
References: Image: http://en.wikipedia.org/wiki/Gel_electrophoresis