Luckau Protocols:Agarose Gel

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

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
 * 3) * 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
 * 4) * Note: make sure all the agarose has dissolved into solution
 * 5) Add 10,000X GelRed when agarose mixture is warm; swirl
 * 6) Allow to cool - do the baby milk test (it should be at a warm temperature, but not too warm for a baby to touch)
 * 7) Pour into caster; set combs and cover with cardboard; allow to harden (10-20 minutes) (will appear cloudy)
 * 8) 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
 * 2) * if loading 6µL into gel, mix 1µL gel load dye with 5µL sample in parafilm
 * 3) Transfer from parafilm into gel wells
 * 4) 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
 * 3) * black gets plugged into the ground
 * 4) * red gets plugged into the red
 * 5) Turn power supply to 'on'
 * 6) Adjust voltage according to table
 * 7) * use the right-side dial to adjust the right-side meter (red numbers)
 * 8) Check for the curtain of small bubbles to ensure everything is working properly
 * 9) Cover with cardboard; takes about 30 min

Image Agarose Gel

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

Migration of gel load dye & Ladder Map

 * Bromophenol Blue (175bp) and Xylene Cyanol FF (1500bp)
 * [[Image:AgaroseGel_2_XCBPB.jpg]]    -       [[Image:1kb Gel Ladder Map.jpg|300 px]]

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]]  [[Image:OwlA2Large_draw.jpg|300 px]]