Luckau Protocols:Agarose Gel

Agarose Gel Protocol

Tara K. Luckau's Home Page Conservation Genetics Lab Notebook Tara's Protocols NanoDrop Agarose Gel Low TE Tris-Cl KCl

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 Gel Red is added during the agarose-making process. The dye binds to the DNA and flouresces when exposed to UV light.

Materials To Be Familiar With

• Gel Rig, caster tray, combs

Protocol

• Use 1% for genomic DNA, 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 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; 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. In titer tray, mix sample with gel loading buffer in 1:6 ratio
   • if loading 6µL into gel, mix 1µL gel loading buffer with 5µL sample in titer tray
2. Transfer from titer tray into gel wells

Run Agarose Gel

1. Attach rig's safe lid and ensure power supply leads fit snugly
2. Plug loose ends of lead into power supply
   • black gets plugged into the ground
   • red gets plugged into the red
3. Turn power supply to 'on'
4. Adjust voltage according to table
   • for most applications 120V should be sufficient
5. Check for the curtain of small bubbles to ensure everything is working properly
6. Takes about 30 min

Image Agarose Gel

1. Assemble kit:
   • Gel (keep on caster)
   • Kim Wipes
   • CompactFlash card
2. Gel Imager (UV transilluminator) is in room 409
3. Power on (top right); plug in CompactFlash card
4. Place gel on glass top; center using 'Live' view
5. Close door; turn on UV light
6. Adjust zoom, focus, aperture and exposure if 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 on box
7. Press save to save image to CompactFlash card; feel free to take multiple images of different settings or zooms

FYI

• Migration of gel loading dyes Bromophenol Blue (175bp) and Xylene Cyanol FF (1500bp)
• 

• To make 1L of 1x TAE from 50x TAE:
• 20mL 50x TAE + 980 NanoPure H₂O