Gel Electrophoresis.

Gel Electrophoresis- “A method that separates macromolecules-either nucleic acids or proteins-on the basis of size, electric charge, and other physical properties. Gel electrophoresis refers to the technique in which molecules are forced across a span of gel, motivated by an electrical current. Activated electrodes at either end of the gel provide the driving force. A molecule's properties determine how rapidly an electric field can move the molecule through a gelatinous medium.”

Plasmid-“Small, circular, extrachromosomal DNA molecules. They can replicate independently of the genome, and are found in numbers ranging from one per cell to hundreds per cell (this is called "copy number"). Plasmids frequently carry genes for antibiotic resistance. While antibiotic resistance is becoming an increasingly important problem medically, it is a useful marker in Recombinant DNA technology. Such markers, along with the small size and potentially high copy number, make plasmids indispensible tools in Molecular Biology”

Restriction Enzymes- “Also known as Restriction Endonucleases, these enzymes recognize and cut specific sequences in double-stranded DNA. Discovered in bacteria in 1962, Restriction Enzymes are made to protect the bacteria from foreign DNA. Bacteria have a method of marking their own DNA as being "self" (called a Modification System). Any DNA not recognized as self is digested into smaller pieces by the Restriction Enzymes. Restriction Enzymes search for exact sequences of a defined length. Some enzymes recognize sequences 4 bp long (e.g., GTAC), some 6 (e.g., GAATTC), and still others 8 or more. One of the common features of most enzyme recognition sites is that they are palindromes. A palindrome is a sequence which is read the same on both strands in the 5' --> 3' direction.”

How to setup a gel electrophoresis
- mix agarose with TBE and heat until solution turns liquid

- pour hot liquid into a casting tray which also has a comb already inserted to make wells after the liquid has cooled down and turned into a gel 

- after digesting the DNA with a restriction enzyme, dyes, which are charged molecules, are also added

- that solution is then transferred into the wells of the gel

- an electric current gets passed through the gel

DNA is a negatively charged molecule because the phosphate groups on the DNA are negatively charged.  This causes the DNA to move towards the positive electrode.  The 2 criterias that determine the rate of movement of DNA fragments are: the magnitude of the charge on the DNA and the size and shape of the DNA.

 

Naming Restriction Enzymes

- “the enzyme name begins with the first letter of the genus of the source the next two letters are the first two letters of the species of the source, the letter after may be a strain designation if needed the name ends in a roman numeral: I if the organism has only one; I and II if there are 2, etc.”

HindII

- “H” stands for the genus Haemophilus 

- “in” stands for the species influenzae

- “d” stands for the strain Rd

- “II” indicates that it was the second enzyme that was isolated from the strain 

- restriction site: 5’ GT(Py)(Pu)AC 3’

                   3’ CA(Pu)(Py)TG 5’

PstI

- first restriction enzyme to be isolated from Providencia stuartii

- restriction site: 5’ CTGCAG 3’

                   3’ GACGTC 5’

HpalI

- firt restriction enzyme to be isolated from Haemophilus parainfluenzae

- restriction site: 5’ GTTAAC 3’

                   3’ CAATTG 5’

SspalI

- first restriction enzyme to be isolated from Sphaerotilus species

- restriction site: 5’ AATATT 3’

                   3’ TTATAA 5’

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