Gel Electrophoresis By Zainab sajjad 117114 Ayesha Rehman

Gel Electrophoresis By Zainab sajjad (117114) Ayesha Rehman (117115)

What is gel electrophoresis § Gel electrophoresis is a laboratory method used to separate mixtures of DNA, RNA, or proteins according to molecular size. § In gel electrophoresis, the molecules to be separated are pushed by an electrical field through a gel that contains small pores.

§ The molecules travel through the pores in the gel at a speed that is inversely related to their lengths. § This means that a small DNA molecule will travel a greater distance through the gel than will a larger DNA molecule.

History § The history of electrophoresis begins in earnest with the work of Arne Tiselius in the 1930 s, and new separation processes and chemical analysis techniques based on electrophoresis continue to be developed into the 21 st century.

gel electrophoresis used for? § Gel electrophoresis is used to test for genes related to specific illnesses, to identify DNA associated with crime scenes, to discover paternity and to track evolutionary relationships. § This technique is important in medical, biological and criminal science.

Purpose of Gel Electrophoresis q. A method for separating DNA q. Can be used to separate the size of q. DNA q. RNA q. Protein

DNA § We start with: A variety of different fragments of DNA all mixed together § We will use gel electrophoresis to separate/sort these fragments

How It Separates § The gel is a porous How It Separates matrix (like a sponge) –Separates DNA based on –Size –Charge

Separation by Size § As DNA is moved through the gel, smaller sized fragments move through faster than larger sized fragments § Ex. A 100 base pair fragment will move through the gel faster than a 500 bp fragment

Separation Using Charge § The charge on DNA is what makes it move through the gel – DNA is a charged molecule. What is the charge on DNA? – Negative charge – Why? – Phosphate group is negatively charged

Separation Using Charge DNA is loaded into the gel on the cathode (-) end § Gel is placed in a buffer solution that will conduct electricity § Electric current is run through the gel § DNA is attracted to the + end (anode)

The Gel § Wells are created to put the DNA into § We use agarose gels to separate DNA

Solution #1 § Problem #1: How can we see the DNA sample as we load it into the gel § Problem #2: How can we make sure DNA won’t float away § Solution: Add loading dye to the initial DNA sample!

Loading Dye § Adds mass to the DNA sample so that it will go into the well § makes it sink to the bottom § Adds blue color so we can see what we are pipeting

Solution #2 § Problem: DNA is colorless. Once the DNA has been run through the gel, how can we see where it is on the gel? § Solution: Add Ethidium Bromide (Et. Br) or Gel Red to the gel

Ethidium Bromide § The DNA intercalates with the Ethidium Bromide (Et. Br) § Intercalates = inserts itself between bases § Gel Red also stains nucleic acids § Et. Br and Gel Red will fluoresce under UV light

Relative Size vs. Absolute Size § In gel, we can determine which fragments of DNA are bigger than others § Relative Size § Which fragment is bigger, A or B § Fragment A (didn’t travel as far in a fixed amount of time)

Absolute Size § How can we determine the actual size of the § § DNA fragments (how many base pairs- bp)? Use a size standard Also called a DNA ladder Consists of a series of fragments of known sizes Use it to compare to our DNA fragments