21.5 gel electrophoresis

What is gel electrophoresis

A technique used to separate molecules based on their size using an electric current applied to agarose gel matrix

What molecules is gel electrophoresis used to separate

DNA, RNA, proteins 

what electrode to DNA and RNA move to

travel down to positive electrode because they are negatively charged - due to negatively charged phosphate group

what electrode do proteins move to

proteins consist of amino acids with different r groups (which may have positive, negative or neutral)

- to standardise their charge (so they all move in same direction) they are denatured with a detergent > causes all proteins to become negatively charged > travel down to positive electrode

why is gel electrophoresis to analyse proteins useful in clinical settings

enables doctors to identify presence of abnormal proteins in e.g. urine, blood to diagnose diseases

How do you use gel electrophoresis apparatus for DNA

1. Place agarose gel matrix (solidified gel with wells) into a gel tank - ensuring wells are at neg. electrode end 

2. Pour buffer solution over gel - to maintain a constant pH & allow conduction of electricity 

3. Mix the sample e.g. DNA with loading dye

4. Use micropipette to add equal volumes of sample into wells & add a DNA ladder to one well - ensure micropipette isnt pushed all the way down to the bottom of the gel  

5. Voltage is applied across gel and DNA fragments move down towards positive electrode

Step 1 - why do the wells have to be at the negative electrode end

Since DNA is negative charged starting at the negative electrode means its repelled to positive electrode

what are the negative and positive electrodes also known as

negative electrode = cathode (attracts pos ions cations)

positive electrode = anode (attracts neg ions anions)

Step 2 - why is constant pH important 

Step 2 - what is conducting electricity 

-To ensure dna fragments stay negatively charged and don’t denature 

-allowing current to pass through

Step 3 - Why is loading dye used rather than just regular dye 

Makes the sample denser so it sinks to bottom of the well to make it visible 

Step 4 - what is a dna ladder 

Step 4 - why is it important micropipette isn't pushed all the way down to bottom of the gel

-a sample of DNA with known lengths which acts like a ruler to compare and find the length of the target DNA fragments 

-pushing it all the way to bottom of gel will damage it 

Step 5 - what fragments move faster through the gel

Agarose gel acts like a mesh allowing smaller fragments to move faster and further down since they move through the pores more easily whereas larger fragments are slowed down 

Outline how DNA is then visualized

1. Switch off voltage and remove gel from tank 

2. Alkaline solution - breaks down hydrogen bonds between complementary base pairs  releasing the single stranded DNA  

3. Southern blotting - transfer single stranded DNA from fragile gel to sturdy nylon membrane to make it easier for analysis 

4. Hybridisation - add radioactive or fluorescent DNA probes which are complementary to the DNA regions

5. Visualisation - use x ray (for radioactive probes) or uv light (for fluorescent probe) to reveal a barcode like pattern of DNA bands