Gel Electrophoresis

1. Gel Electrophoresis

2. Purpose of Gel Electrophoresis • A method for separating DNA • Can be used to separate the size of • DNA • RNA • Protein • We will be using it to separate DNA

3. DNA • What you start with: A variety of different fragments of DNA all mixed together • We will use gel electrophoresis to separate/sort these fragments

4. How It Separates • The gel is a porous matrix (like a sponge) • Separates DNA based on • Size • Charge

5. start end 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 Image taken without permission from http://www.dnai.org/b/index.html-- Gel Electrophoresis Animation

6. 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 Image taken without permission from http://www.dnai.org/b/index.html-- Gel Electrophoresis Animation

7. Separation Using Charge • The gel is hooked up to a power source • 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) = “runs to the red” Image taken without permission from http://www.dnai.org/b/index.html-- Gel Electrophoresis Animation

8. wells - well Direction DNA travels - + Direction DNA travels SIDE VIEW TOP VIEW + The Gel • Wells are created to put the DNA into • We use agarose gels to separate DNA

9. Challenges • DNA is colorless-- how will we see it on the gel & when we are loading it into the gel? • How do we get the DNA to stay in the well (not float away)?

10. 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!

11. 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 you can see what you are pipetting

12. 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 (EtBr) or Gel Red to the gel

13. Ethidium Bromide • The DNA intercalates with the Ethidium Bromide (EtBr) • Intercalates = inserts itself between bases • GelRed also stains nucleic acids • EtBr and GelRed will fluoresce under UV light

14. (-) start A B (+) end Relative Size vs. Absolute Size • Looking at a gel, you 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)

15. 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 your DNA fragments

16. 1000 bp 850 bp 750 bp 600 bp 200 bp 100 bp Example Size Standard Sample 1 Sample 2 - • Suppose you have a size standard with the following sized fragments: 1000 bp, 850 bp, 750 bp, 600 bp, 200 bp, 100 bp • Based this info, how big is the circled fragment? • 850 bp +