Gel ElectrophoresisGel Electrophoresis

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Zainab sajjad (117114)Zainab sajjad (117114)

Ayesha Rehman (117115)Ayesha Rehman (117115)

What is gel electrophoresisWhat is gel electrophoresis

Gel electrophoresis is a laboratory Gel electrophoresis is a laboratory method used to separate mixtures of method used to separate mixtures of DNA, RNA, or proteins according to DNA, RNA, or proteins according to molecular size.molecular size.

In gel electrophoresis, the molecules In gel electrophoresis, the molecules to be separated are pushed by an to be separated are pushed by an electrical field through a gel that electrical field through a gel that contains small pores. contains small pores.

The molecules travel through the The molecules travel through the pores in the gel at a speed that is pores in the gel at a speed that is inversely related to their lengths. inversely related to their lengths.

This means that a small DNA This means that a small DNA molecule will travel a greater molecule will travel a greater distance through the gel than will a distance through the gel than will a larger DNA molecule. larger DNA molecule.

HistoryHistory The history of electrophoresis begins The history of electrophoresis begins

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

gel electrophoresis used for?gel electrophoresis used for?

Gel electrophoresis is used to test for Gel electrophoresis is used to test for genes related to specific illnesses, to genes related to specific illnesses, to identify DNA associated with crime identify DNA associated with crime scenes, to discover paternity and to scenes, to discover paternity and to track evolutionary relationships. track evolutionary relationships. 

This technique is important in This technique is important in medical, biological and criminal medical, biological and criminal science. science.

Purpose of Gel ElectrophoresisPurpose of Gel Electrophoresis

A method for separating DNA A method for separating DNA Can be used to separate the size ofCan be used to separate the size ofDNARNAProtein

DNADNA

We start with: A variety of We start with: A variety of different fragments of DNA all different fragments of DNA all mixed togethermixed together

We will use gel electrophoresis We will use gel electrophoresis to separate/sort these to separate/sort these fragments fragments

How It Separates How It Separates

The gel is a porous How It Separates The gel is a porous How It Separates matrix (like a sponge) matrix (like a sponge)

–Separates DNA based onSeparates DNA based on

–Size

–Charge

Separation by Size Separation by Size

As DNA is moved through the As DNA is moved through the gel, smaller sized fragments gel, smaller sized fragments move through faster than larger move through faster than larger sized fragments sized fragments

Ex. A 100 base pair fragment will move through the gel faster than a 500 bp fragment

Separation Using Charge Separation Using Charge

The charge on DNA is what makes it The charge on DNA is what makes it move through the gelmove through the gel– DNA is a charged molecule. What is DNA is a charged molecule. What is

the charge on DNA?the charge on DNA?– Negative charge– Why?Why?– Phosphate group is negatively

charged

Separation Using Charge Separation Using Charge

DNA is loaded into the gel on the cathode (-) DNA is loaded into the gel on the cathode (-) endend

Gel is placed in a buffer solution that will Gel is placed in a buffer solution that will conduct electricityconduct electricity

Electric current is run through the gelElectric current is run through the gel DNA is attracted to the + end

(anode)

The GelThe Gel

Wells are created to put the DNA intoWells are created to put the DNA into We use agarose gels to separate We use agarose gels to separate

DNADNA

Solution #1 Solution #1

Problem #1: How can we see the Problem #1: How can we see the DNA sample as we load it into the gelDNA sample as we load it into the gel

Problem #2: How can we make sure Problem #2: How can we make sure DNA wonDNA won’t float away ’t float away

Solution: Add loading dye to the Solution: Add loading dye to the initial DNA sample!initial DNA sample!

Loading Dye Loading Dye

Adds mass to the DNA sample so that it Adds mass to the DNA sample so that it will go into the wellwill go into the well

makes it sink to the bottom Adds blue color so we can see what we Adds blue color so we can see what we

are pipetingare pipeting

Solution #2 Solution #2

Problem: DNA is colorless. Once the Problem: DNA is colorless. Once the DNA has been run through the gel, DNA has been run through the gel, how can we see where it is on the how can we see where it is on the gel?gel?

Solution: Add Ethidium Bromide Solution: Add Ethidium Bromide (EtBr) or Gel Red to the gel(EtBr) or Gel Red to the gel

Ethidium Bromide Ethidium Bromide

The DNA intercalates with the The DNA intercalates with the Ethidium Bromide (EtBr) Ethidium Bromide (EtBr)

Intercalates = inserts itself between bases

Gel Red also stains nucleic acidsGel Red also stains nucleic acids EtBr and Gel Red will fluoresce EtBr and Gel Red will fluoresce

under UV lightunder UV light

Relative Size vs. Absolute Relative Size vs. Absolute Size Size In gel, we can determine which In gel, we can determine which

fragments of DNA are bigger than fragments of DNA are bigger than othersothers

Relative SizeRelative Size Which fragment is bigger, A or BWhich fragment is bigger, A or B Fragment A (didn’t travel as far in a

fixed amount of time)

Absolute Size Absolute Size

How can we determine the actual size of How can we determine the actual size of the DNA fragments (how many base pairs- the DNA fragments (how many base pairs- bp)?bp)?

Use a size standardUse 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