1 / 16

Gel Electrophoresis

Gel Electrophoresis. 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. DNA . What you start with: A variety of different fragments of DNA all mixed together

hedy
Download Presentation

Gel Electrophoresis

An Image/Link below is provided (as is) to download presentation Download Policy: Content on the Website is provided to you AS IS for your information and personal use and may not be sold / licensed / shared on other websites without getting consent from its author. Content is provided to you AS IS for your information and personal use only. Download presentation by click this link. While downloading, if for some reason you are not able to download a presentation, the publisher may have deleted the file from their server. During download, if you can't get a presentation, the file might be deleted by the publisher.

E N D

Presentation Transcript


  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 +

More Related