Understanding Gel Electrophoresis Fill in more on digests 9/20/2018

Overview of topics covered Basic Principles Demonstration of a gel Ladders as references Optimizing the separation 9/20/2018

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 Now it would be great to make a picture of many lines of various sizes mixed up on one size and organize according to size in the other 9/20/2018

Basic Principle of Electrophoresis A mixture of DNA molecules becomes organized by size 9/20/2018

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 9/20/2018

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 9/20/2018

- - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - + + + + + + + + + + + + + + + + + + + + + + + + ++ + + + + + + + + + DNA is negatively charged and therefore repelled from the negative pole and attracted towards the positive pole 9/20/2018 - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - -

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. 9/20/2018

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A Typical Electrophoresis Gel Setup Negative end - - - - - - - - - - - - - Direction of DNA movement Direction of DNA movement Positive end + + + + + + + + + + 9/20/2018

A Typical Image of an Agarose Gel Under UV Light Largest DNA fragments Decreasing DNA Size Smallest DNA fragments The DNA fragments can be visualized using a special dye that specifically binds DNA and fluoresces under illumination with UV light 9/20/2018

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 9/20/2018

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 9/20/2018

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 9/20/2018

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) Put in image of a 100 and 1000 bp ladders on a gel- is it possible given the size- Do I need to explain the relative nature of the ladders – they will always line up in the same order but not always the same distance Image modified from http://www.invitrogen.com/images/lrgegeldnamarkers.jpg 9/20/2018

Sizing a Gel Product 2000 bp 1000 bp ladder Base Pairs (bp) 4000 3000 1600 1000 500 2000 bp 1000 bp Put in an image of a ladder and a sample with arrows indicating the sizes 1Kbp Sample ladder 9/20/2018

What is the Size of this Fragment? 4000 3000 2000 1600 1000 500 Image of one fragment against a ladder- this first one should have arrows with the scale and the band in the middle- they have to get the concept that one can read in between Sample 1Kbp ladder 9/20/2018

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 9/20/2018

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 9/20/2018

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 9/20/2018

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 9/20/2018

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. 9/20/2018

What is the Size of this Fragment? The 100 bp ladder was used Image of a sample with the 100 bp ladder used and the sample Reference Ladder 9/20/2018

The size of the fragment is… Size In Base Pair (bp) 1500 1200 1000 600 500 400 300 200 ≈175 bp 100 9/20/2018

What is the Size of these Fragments? The 1 kbp ladder was used 4000 3000 2000 1600 1000 500 9/20/2018

The size of the fragments are… Sizes in Base Pairs (bp) 5000 4000 3000 2000 ≈1800 bp 1600 1000 ≈1100 bp 500 9/20/2018

What is the Size of these Fragments? The 1 kbp ladder was used 4000 3000 2000 1600 1000 500 Image of a sample with the 100 bp ladder used and the sample . At this point I think they should get an exercise sheet with various sample gels and they must size the products 9/20/2018

The Size of the fragments are… 6000 bp 5000 4000 3000 ≈1800 bp 2000 1600 ≈1100 bp ≈800 bp 1000 500 9/20/2018

Optimizing Your Gel Electrophoresis One parameter that can be altered to optimize a gel electrophoresis is the agarose concentration in the gel 9/20/2018

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 Image of spaghetti and a molecule at the front of the mess- arrow pointing to DNA and arrow to spaghetti as chains of agarose 9/20/2018

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 9/20/2018

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 9/20/2018

Effect of Percent Agarose on Fragment Separation An 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 500 400 bp 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 9/20/2018