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Teacher's Guide: Techniques Lab C-2,
Casting and Loading an Agarose Gel
Separation of Assorted Charged Dyes

This lab is designed to be a follow-up to C-1 for the adventurous and/or advanced student. It can also be used for CHEMISTRY classes.

Time of lab
  • One FULL period. Explain lab procedures the day before you do the lab.

Preparation before lab
  • Mix one batch of 0.8% agarose in TAE or TBE buffer (not water!). For example, 400 mL of 0.8% agarose would be enough for 20 teams each using 20-25 mL. To make this, mix 3.2 g of agarose power in 400 mL of 1X TAE buffer. SEE TECH TIPS, BELOW.

  • If you are using agarose sent from previous schools, keep an eye out for 2 possible problems:
    1. Wrong concentration agarose. If much less than 0.6%, gels will be flimsy and break easily. If much greater than 1%, dyes will migrate more slowly and not separate very much in the time allotted.
    2. Improper buffer concentration. Gels made in water will solidify clearer than gels made with buffer. Dyes may move into adjacent wells. Dyes will be smeary if run 20+ minutes. Check pH if suspect water. Gels made in higher than 1X buffer will heat up more quickly and dyes will be smeary. Check current for clue about buffer concentration.

  • Check dyes. You'll need 2 µL per team. Dyes have been dissolved in 10% glycerol, 10mM Tris, 1 mM EDTA, pH 8. The concentration of dyes is approximately 0.5%. You'll want to be sure each team loads xylene cyanole and bromphenol blue, the dyes in our "loading dye" used to track DNA in a gel. XC is a negatively charged dye that migrates slower than most DNA: BPB is a negatively charged dye that migrates faster than the DNA sizes we use. Glycerol is used to increase the density of the loaded sample so that the sample sinks down into the wells.

    Below is the list of dyes provided by the Gene Connection to it's schools.
Dye/pH indicator Charge Formula weight Other info
Bromcresol purple Neg    
Bromcresol green Neg    
Bromthymol blue Neg 624 yellow at pH 6, blue at pH 7.6
Bromphenol blue* Neg 670 yellow at pH 3, blue at pH 4.6
Xylene cyanole* Neg 539  
Orange G Neg 452 red at pH 1.4, yellow at pH 2.6
Congo red Neg 697  
Phenol red Neg 354 yellow at pH 6.8, red at pH 8.4
Brilliant cresyl blue Pos 386 (maybe 2 colors)
Brilliant green Pos 483  
Methylene green Pos    
Methylene blue Pos 320  
Methylene violet Pos    

*Note: migration by these dyes may be more influenced by charge than by size.

  • Students could choose (a) a random assortment of dyes, (b) all blue or green or red dyes, (c) all dyes with the same prefix, i.e., "methylene" or "brom". In the latter two cases, it is important for them to predict charge... will all blue dyes be charged alike? or will all dyes with "brom" have the same charge?

  • 1X TAE or TBE carboys or jugs (for filling up gel boxes) should be set out in the classroom. To expedite dispensing of buffer, provide several beakers near the carboys.

  • Only the first class of the day needs to fill gel boxes. 1X TAE or TBE will "last" for an entire day; it should NOT be discarded at the end of each period! Gently slosh the boxes to mix the ions...

  • Nothing needs to be sterile for this lab.

What to demonstrate/explain
  • Show how to raise or lower, and secure, the "gates" on the ends of the casting trays. Unless you tell them not to, students may remove the screws entirely. They need only be loosened. Students might also over-tighten the screws. Screws should be adjusted so that the gates are snug with the edges of the tray, yet the gates can be moved up and down with reasonable effort. Once the tension on the gates has been properly adjusted, it is not usually necessary to loosen and re-tighten the screws with each use.

  • Review how to properly slide off gel box lid. DO NOT PULL ON WIRES!!!

  • Review how to use pipet for tiny volumes of viscous solutions (Lab A ext).

  • Demonstrate how to load a well without "puncturing" the bottom of the gel with the pipet tip.

  • Careful -- loading dye tubes "splatter" as they are opened. The dye stains hands and clothes.


Tech Tips
  • Dissolve (or re-liquefy) agarose by microwaving. It takes only 5-6 min. on MEDIUM to liquefy a 500-mL bottle of agarose. THE LID MUST BE LOOSE. After heating, do not swirl too vigorously, or superheated agarose can bubble out of the bottle.

  • HOLD melted agarose in a 65°C water bath for use during the period. Students dispense what they need (~20-25 mL), insuring that the agarose will not be so hot that it warps the casting trays as it is poured.

  • Our choice of 20-25 mL is a trade-off between gel running time (thinner runs faster) and gel durability (thicker gels are easier to handle without tearing).

  • Students must record their results immediately upon completion of electrophoresis. These dyes will diffuse rather quickly.

  • The 1X TAE (or TBE) buffer can be reused about 5 times--check current before replacing.

Post Lab
  • At the end of the day, after all classes have completed the lab, the 1X TAE or TBE and the blue agarose can be discarded.

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