3B 1012852 Mode D'emploi page 4

that you need. To dissolve the agarose, heat the Erlenmeyer flask either in a microwave or with a heating stirrer. For the
latter, use medium heating power and stir constantly. Heating in a microwave should only be for short periods of time, on
medium heating level, repeated multiple times. When heating in a microwave, remove the Erlenmeyer flask from time to
time (remember to wear gloves, goggles and be careful to avoid boiling!) and gently swirl the gel solution in a circle. Put it
back in the microwave afterwards and repeat the entire process 3-4 times, until the agarose is completely dissolved.
4. B efore the casting of the gel, allow the gel solution to cool down to 60°C. Cast the gel solution without any air bubbles in
the gap between the two white plastic stripes on the chamber's ground (its capacity is about 40 ml). Then insert the comb
(or both combs respectively). Make sure that there are no air bubbles on the edges of the slots. Gels made of standard aga-
rose solidify within 20 minutes at room temperature.
5. A fter the solidification of the agarose gel, add some of the electrophoresis buffer in 3-mm layers in the chamber over the
gel to prevent i from drying out. After carefully removing the comb(s), the gel can now be loaded, The coated gel can be
stored for a few days at 4°C. For best results leave the comb inserted and cover the gel with wrapping film to protect it
from drying out.
Loading of gels and electrophoresis
1. A fter the solidification of the agarose gel, cover the gel with the electrophoresis buffer. Please note the maximum filling
level for the electrophoresis buffer.
2. Remove the comb(s) from the agarose gel by gently moving back and forth and then carefully pulling out upright.
3. I n the next step, the slots in the gel can be loaded with the samples. Preparing the samples with an appropriate gel loading
buffer which increases the specific weight of the samples makes it easier to pour them into the slots with a micropipette
(see section "gel loading buffer").
4. W hen loading the gel slots, dip the tip of the micropipette carefully into the slot and then slowly press out the sample.
Make sure not to damage the bottom of the slot.
Beginners should practise this with „practice samples" consisting of H2O and gel loading buffer only.
5. At least one size standard should be applied on each gel so that the size of the various DNA fragments can be determined.
6. N ow place the safety lid on the electrophoresis chamber and connect the chamber to a suitable power supply.
P lease note the correct polarity. In an alkaline up to a neutral medium, nucleic acids are charged negatively and migrate to
the anode (red pole).
7. T urn on the power supply and run the electrophoresis at a voltage in the range 80 - 120 V. The process of the electrophore-
sis can be observed with the help of the dye, which is inside the gel loading buffer.
8. B romophenol blue and xylene cyanol are frequently used dyes for the gel loading buffer. These dyes are also negatively
chargedand migrate to the anode. This process, the so-called co-migration with the double-stranded DNA fragments,
depends on different factors, such as agarose type, electrophoresis buffer and gel strength.
F or a rough estimation: bromophenol blue migrates in 1xTAE electrophoresis buffer and 1%-standard agarose gel in the
same way as a DNA fragment of 650 base pairs. Under the same conditions, xylene cyanol migrates like a fragment with
5 000 base pairs.
Staining of nucleic acids
Since there are different methods of staining nucleic acids, we refer at this point to the corresponding technical literature
(Sambrook et. al., 1989).
Evaluation / documentation
Size determinations of nucleic acid fragments can be carried out by comparison with the fragments of a length standard. A
detailed description of this procedure can be found, for example, in Molecular Genetics by R. Knippers (2008).
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