Luckau Protocols:Agarose Gel

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Agarose Gel Protocol

Tara K. Luckau's Home Page

Conservation Genetics Lab Notebook

Tara's Protocols

Contents

Purpose

Agarose gels are used to verify the presence and estimate the size of DNA (both genomic and amplified). When loaded into a gel and subjected to an electric current, DNA fragments migrate to the positive terminal. Small DNA fragments migrate more rapidly than large ones. By running a commercially available ladder of fragments of known size, we can estimate the DNA fragment size in our sample of interest.

To visualize the DNA after electrophoresis, a dye called GelRed is added during the agarose-making process. The dye binds to the DNA and flouresces when exposed to UV light.


Protocol

Use 1.5% for genomic DNA; use 2% for amplified DNA

Gel Rig Size 1x TAE (mL) 1.5% agarose (g) 2% agarose (g) Gel Red (µL) Sample volume (µL) max voltage (V)
small (10cm x 10cm) 50 _ 1 5 4-6 80
medium (x x) 130 _ 2.6 13 24-tooth comb: 6-10 120
36-tooth comb: 3-5
large (20cm x 27cm) 270 _ 5.4 27 24-tooth comb: 6-10 170
36-tooth comb: 3-5


Pour Agarose Gel

  1. According to the table above, mix agarose powder and 1x TAE in Erlenmeyer flask; swirl
  2. Microwave on 1/2 power until the mixture starts to boil
    • Note: be careful not to let the mixture boil over, or you'll lose much of your volume and will have a mess to clean up
    • Note: make sure all the agarose has dissolved into solution
  3. Add 10,000X GelRed when agarose mixture is warm; swirl
  4. Allow to cool - do the baby milk test (it should be at a warm temperature, but not too warm for a baby to touch)
  5. Pour into caster; set combs and cover with cardboard; allow to harden (10-20 minutes) (will appear cloudy)
  6. Remove combs
  • Pre-poured gels may be stored covered in the fridge for up to 1 week


Load Agarose Gel

  1. On parafilm, mix sample with gel load dye in 1:6 ratio
    • if loading 6µL into gel, mix 1µL gel load dye with 5µL sample in parafilm
  2. Transfer from parafilm into gel wells
  3. Load same volume (6µL) of ladder into wells on either side of your samples


Run Agarose Gel

  1. Attach rig's safe lid and ensure power supply leads fit snugly
  2. Plug loose ends into power supply, as marked
    • black gets plugged into the ground
    • red gets plugged into the red
  3. Turn power supply to 'on'
  4. Adjust voltage according to table
    • use the right-side dial to adjust the right-side meter (red numbers)
  5. Check for the curtain of small bubbles to ensure everything is working properly
  6. Cover with cardboard; takes about 30 min


Image Agarose Gel

  1. Assemble cafeteria tray:
    • Gel (keep on caster)
    • Kim Wipes
    • USB stick
    • gloves
    • room key (in drawer)
  2. Gel Imager (UV transilluminator) is in room 215
    • CAUTION! Ethidium bromide (EtBr) is a carcinogen, mutagen and teratogen, and it's used (instead of GelRed) by other users of this room.
    • Keep one hand gloved to handle EtBr stuff, one hand ungloved to handle clean stuff
    • The gel imager is EtBr-contaminated; the computer is CLEAN!
  3. Plug in USB drive; open the program "AlphaImager Mini"
  4. Place gel on glass top; center and zoom
  5. Close door; turn on UV light (bottom right)
  6. Adjust zoom, focus, aperture and exposure as needed
    • Zoom: middle knob on camera (which is mounted on top of the box)
    • Focus: bottom knob on camera
    • Aperture: top knob on camera
    • Exposure: arrow buttons in computer program
  7. Click 'Acquire' then 'Save'
    • save as .jpg (not .tif) to the USB stick
    • feel free to take multiple images of different settings or zooms


FYI

Migration of gel load dye & Ladder Map

  • Bromophenol Blue (175bp) and Xylene Cyanol FF (1500bp)
Image:AgaroseGel_2_XCBPB.jpg -------------


To make 1L of 1x TAE from 50x TAE

20mL 50x TAE + 980 NanoPure H2O

Materials To Be Familiar With

  • Gel Rig, caster tray, combs
Image:OwlA2Large.jpg
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