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

FYI:

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