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Understanding Gel Electrophoresis. Overview of topics covered. Basic Principles Demonstration of a gel Ladders as references Optimizing the separation. Gel electrophoresis separates a mixture of DNA fragments according to size

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Presentation Transcript
overview of topics covered
Overview of topics covered
  • Basic Principles
  • Demonstration of a gel
  • Ladders as references
  • Optimizing the separation
purpose of gel electrophoresis
Gel electrophoresis separates a mixture of DNA fragments according to size

This is accomplished by drawing the DNA fragments through a gel

Purpose of Gel Electrophoresis
slide4

Basic Principle of Electrophoresis

A mixture of DNA molecules becomes organized by size

how does gel electrophoresis separate dna fragments
How does gel electrophoresis separate DNA fragments?
  • The gel acts as a sieve to filter the DNA fragments
  • The DNA fragments are naturally negatively charged due to the phosphate backbone
  • DNA fragments of differing sizes will move though the gel at differing rates
  • Smaller fragments move faster through the gel and larger fragments move slower
the need for an electric field
The Need for an Electric Field
  • The electrostatic charges set up in the gel act as the “force”
  • The anode has a relative lack of electrons and has a net positive charge
  • The cathode is rich in electrons and has a net negative charge
  • The DNA is loaded in wells near the cathode
  • The DNA is pulled towards the anode by electric attraction
slide7

+ + + + + + + + + + + + + + + + + + + + + + + + ++ + + + + + + + + +

DNA is negatively charged and therefore repelled from the negative pole and attracted towards the positive pole

- - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - -

size selection in an agarose gel
Size selection in an Agarose Gel
  • The agarose gel is full of tiny pores which the DNA can slide through. The smaller pieces can easily find a route to the positive terminal.
  • The larger fragments cannot slide through the pores as easily.
  • This results in the smaller fragments moving quickly to the positive end of the gel and the larger fragments move much slower and remain close to the negative end of the gel.
slide9

+ + + + + + + + + + + + +

_ _ _ _ _ _ _ _ _ _ _ _

a typical electrophoresis gel setup
A Typical Electrophoresis Gel Setup

Negative end- - - - - - - - - - - - -

Direction

of DNA

movement

Direction

of DNA

movement

Positive end+ + + + + + + + + +

a typical image of an agarose gel under uv light
A Typical Image of an Agarose Gel Under UV Light

Largest DNA fragments

Decreasing

DNA

Size

  • The DNA fragments can be visualized using a special dye that specifically binds DNA and fluoresces under illumination with UV light

Smallest DNA fragments

the intensity of the band is proportional to the concentration of dna
The Intensity of the Band is Proportional to the Concentration Of DNA
  • An important point to remember is that the intensity of the band is proportional to the amount of DNA found in the band

The upper band has far less

DNA when compared to the lower

band. The intensity of the bands

are proportional to the amount of DNA at that position in the gel

sizing the fragments of dna
Sizing The Fragments of DNA
  • The sizes of the various fragments can be identified by including a “ladder” in the gel
    • A ladder is a mixture of DNA fragments of known size
    • A ladder is usually run beside the unknown sample so that the sizes of the various DNA fragments in the sample can be identified
ladder
Ladder
  • A ladder is a mixture of DNA fragments of selected sizes
  • When run in a gel electrophoresis, these fragments will separate into distinct bands that can be used as references
  • The size of a fragment is always stated as [X] base pairs (bp)
  • Two common ladders are the 100bp and the 1kbp (1000 bp) ladders
  • Ladders are commercially available and purchased from suppliers
typical ladders 100 bp 1 kbp 1000 bp
Typical Ladders-100 bp & 1 kbp (1000 bp)
  • The 100 bp ladder is composed of a mixture of small fragments (100 to 2000 bp)
  • The 1000 bp ladder is composed of a mixture of larger fragments (500 to 12000 bp)
sizing a gel product
Sizing a Gel Product

Base

Pairs

(bp)

4000

3000

2000

1600

1000

500

2000 bp

1000 bp

1Kbp Sample

ladder

what is the size of this fragment
What is the Size of this Fragment?

4000

3000

2000

1600

1000

500

Sample 1Kbp

ladder

the size of this fragment is
The size of this fragment is…

4000

3000

2000

1600

1000

500

Sample 1Kbp

ladder

The fragment is between the 2000 bp and 3000 bp band (in red)

It is approximately 2500 bp in size

reference bands in ladders
Reference Bands in Ladders
  • Ladder scales are not written on a gel but one is given certain characteristic bands to use as landmarks and are easily identifiable

For Example:

    • In the 100 bp ladder, the 500 bp and the 1000 bp bands are made to stand out
    • In the 1 kbp (1000 bp) ladder, the 1600 bp band stands out
the 100 bp ladder
The 100 bp Ladder
  • In the 100 bp ladder the reference bands are the 500 and 1000 bp

(0.5 and 1.0 kbp)

  • Every band above and below increase by 100 bp
the 1 kbp ladder 1000 bp
The 1 kbp ladder (1000 bp)
  • The reference (bright) band is 1.6 kbp
  • The band directly above is 2.0 kbp and increases by 1.0 kbp as you go up
  • Below the reference band lies the 1.0 kbp and the 0.5 kbp bands

1.6 kbp reference band

using a ladder
Using A Ladder
  • To use a ladder for size estimation, one must locate the reference band(s). This is usually one band that is particularly bright.
  • Once you have found this band and know the distribution of fragment sizes of the ladder, one can start estimating the sizes of the samples.
what is the size of this fragment23
What is the Size of this Fragment?

The 100 bp ladder was used

Reference Ladder

the size of the fragment is
The size of the fragment is…

Size In Base Pair (bp)

1500

1200

1000

600

500

400

300

200

≈175 bp

100

what is the size of these fragments
What is the Size of these Fragments?

The 1 kbp ladder was used

4000

3000

2000

1600

1000

500

the size of the fragments are
The size of the fragments are…

Sizes in Base Pairs (bp)

5000

4000

3000

≈1800 bp

2000

1600

≈1100 bp

1000

500

what is the size of these fragments27
What is the Size of these Fragments?

The 1 kbp ladder was used

4000

3000

2000

1600

1000

500

the size of the fragments are28
The Size of the fragments are…

6000 bp

5000

4000

3000

≈1800 bp

2000

1600

≈1100 bp

1000

≈800 bp

500

optimizing your gel electrophoresis
Optimizing Your Gel Electrophoresis

One parameter that can be altered to optimize a gel electrophoresis is the agarose concentration in the gel

background about agarose
Background About Agarose
  • Agarose is a mixture of long chains of saccharides (sugar)
  • It is extracted from a seaweed and is used in oriental cuisine as a gelling agent for deserts, much like gelatin is used in European cuisine
  • The greater the agarose concentration, the smaller the pores within the gel
  • This changes the ease with which the DNA can travel through the gel
effects of changing gel concentration
Effects of Changing Gel Concentration
  • By increasing the agarose concentration the smaller DNA fragments will give a clearer separation
  • By lowering the agarose concentration the larger fragments of DNA will give a clearer separation
  • By optimizing the % agarose one can clearly separate a mixture of similar DNA fragments
effect of percent agarose on fragment separation
Effect of Percent Agarose on Fragment Separation

As stated, the % agarose in the gel affects the size of the pores in a gel, which affects the speed to which DNA can travel through the gel.

Higher percent agarose gels resolving smaller base pair fragments better than low percent agarose gels

High base pair fragments resolve better in low percent agarose gels

effect of percent agarose on fragment separation an example
Effect of Percent Agarose on Fragment SeparationAn example

The 1kbp ladder and the 100bp ladder were run with 0.8 and 1.6 % agarose gels under the same conditions

1600

1000

1600 bp

500

400bp

1000 bp

300 bp

500 bp

200 bp

0.8% Agarose

80V – 2 hours

1.6% Agarose

80V – 2 hours

Note how the bands have moved further in the low % agarose gel and the higher bp fragment are better defined. The low bp fragments are better defined in the high % agarose gel and are unresolved in the low % agarose gel

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