Materials
Electrophoresis Buffer
concentrate, TAE (40X or 50X)
or LB buffer (10X or 20X)
Beakers, 600 mL
Agarose
Balance, tabletop milligram
Weigh boat, 3.5”×3.5”
Lab scoops
Media bottle, 250 mL
Permanent lab marker pens
Microwave oven
Hot hands protector
Glasses, safety
Gel box, horizontal, for agarose
gels
Beakers, 50 mL
Water bath, 65°C
Procedure
• A 0.8% gel is prepared in the following procedures since that is the appropriate
concentration for resolving a variety of DNA fragment lengths.
• Prior to step 1, prepare 500 mL of 1X electrophoresis buffer from the buffer concentrate
provided. The 1X buffer will be used in this and in the following activity. Use the C1 V1 =
C2 V2 equation. Record the calculations and recipe in your notebook.
1. Make 100 mL of 0.8% agarose in 1X electrophoresis buffer solution. Determine the recipe for
a 0.8% gel, below.
0.8% of 100 mL = _________ g of agarose in a total volume of 100 mL of 1X electrophoresis buffer
2. Obtain a clean 250-mL media bottle and cap. Label it.
3. Weigh out the required mass of powdered agarose in a weigh boat. Add it to the media bottle.
4. Measure out enough 1X electrophoresis buffer to prepare a total of 100 mL of agarose and buffer
mixed together. Add the buffer slowly to the agarose in the media bottle, swirling it to mix.
5. Loosely cap the top of the media bottle. Swirl the flask gently to suspend the agarose in the buffer.
6. To dissolve the agarose in the buffer, microwave the suspension for 4 minutes at 50% power.
7. Wearing bottle holders and safety
goggles, lift the beaker toward the
light (but away from your face),
and very slowly and gently swirl
it. Caution: Do not swirl too fast
because it could boil over. Look
to see if all the agarose crystals have
dissolved. Agarose crystals that do
not dissolve will impede molecular
motion through the gel and will
affect electrophoresis results. If any
agarose crystals are still floating
in the buffer, reheat the solution at
50% for 2 minutes more.
8. Using hot hand protectors, place
the hot dissolved agarose solution
on a fireproof lab tabletop.
Figure 4.18. If the agarose solution is not to be used
9. The solution must cool to
immediately, it can be kept hot in a 65°C water bath until it is
approximately 65°C before
time to cast the gels.
pouring it (see Figure 4.18).
Photo by author.
10. Prepare the gel tray found in your
gel box for pouring (casting) a 7mm thick agarose gel.
Note: Different gel boxes have different types of gel-casting trays. Some trays have gates at
the ends that are screwed into an upright position for gel casting. Some gel trays use tape to
seal the ends. Some gel trays have rubber gaskets that seal the ends of the tray in the gel box
for pouring. Your instructor will show you how your gel tray should be set up for gel pouring.
11. When the agarose solution is about 65°C (just barely cool enough to hold), pour about 30 mL
of it into the gel tray. The gel should be about 7mm thick.
12. Quickly place a six-well comb into the notches at the end of the gel tray. Make sure the comb
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