ease limit your answers to a few sentences IN YOUR LAB NOTEBOOK 1. If restrictio
ID: 141226 • Letter: E
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ease limit your answers to a few sentences IN YOUR LAB NOTEBOOK 1. If restriction enzymes are present in cells, why is their own DNA not cleaved as a result? 2. Many restriction enzymes cleave at unique sequences of 4-8 DNA base pairs that are palindromic. What does this mean? What is the difference between "sticky ends" and "blunt ends?" 3. Which type of restriction endonucleases (1, II, or III) are used for in vitro DNA modifications? What enzyme is used to reconnect the bonds broken by these restriction endonucleases? 4. Of the following, which is so temperature sensitive that it must be returned to ice or the freezer after no more than a few seconds? a. Ethidium bromide b. Your plasmid solution c. Molecular weight markers d. Restriction enzymes e. 6X loading buffer f. restriction enzyme buffers (i.e. buffers 2 and 3) What is the purpose of using the 6X running buffer when loading your samples on the electrophoresis gel? How will increasing the agarose percentage affect the velocity o DNA through the gel? Why? .Explanation / Answer
1. Restriction enzymes are enzymes that cleaves DNA at specific recognition sites known as restriction sites. These enzymes prevents the entry of viral DNA into bacterial cells. The bacterial DNA is however protected from cleavage by methyltransferase that adds methyl groups to DNA bases and prevents cleavage of the recognition sites.
2. Palindromic sequences are DNA sequences that reads the same when read from both 5` to 3` direction and 3` to 5` direction. For eg consider the following palindromic sequence:
5` GGATCC 3`
3` CCTAGG 5`
Some restriction enzymes such as EcoR 1 cuts DNA in a staggered manner and produces overhangs. These are known as sticky ends, these protruding single stranded ends anneal to its complementary strands and can easily be ligated.
Whereas blunt ends are created when restriction enzymes cuts straight in the DNA molecule. Blunt ended molecules are difficult to ligate as compared to sticky ends.
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