Luckau Protocols
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Contents |
Tara Luckau Protocols
Clark Laboratory
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 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
- Make gel using 1x TAE and agarose
- 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 | 50 | _ | 1 | 5 | 4-6 | 80 |
| medium | 130 | _ | 2.6 | 13 | 24-tooth comb: 6-10 | 120 |
| 36-tooth comb: 3-5 | ||||||
| large | _ | _ | _ | _ | _ | 170 |
