Loading Of Samples And Electrophoresis - VWR Peqlab PerfectBlue Mini S Instruction Manual

Instruction Manual PerfectBlue

Loading of samples and electrophoresis

1. Once the gel is completely solidified, lift the tray out of the chamber, turn it 90 °, and replace it in
the chamber with the first comb closest to the cathode side (black electrode) of the chamber. The
running position exposes the open ends of the agarose to the buffer.
2. Pour enough compatible running buffer into the unit to fill chamber and completely cover and sub-
merge the gel. A 'Fill Line' is located on each unit to clearly mark the correct buffer level. See
'Technical properties' for approximate buffer volumes needed for your unit. Too little buffer may
cause the gel to dry out during the run, while excess buffer may slow DNA migration in the gel, in-
crease heat build-up and cause distorted bands.
3. Carefully remove the comb (or combs) by tapping lightly to loosen, and slowly lift straight up out of
the gel tray to avoid damage to the wells.
4. Load prepared samples into the wells. Samples should be mixed with a sample loading buffer (giv-
ing weight to the samples so that they drop evenly into the wells), and contain tracking dye to moni-
tor the gel run. For details on approximate well volumes see 'TECHNICAL SUPPORT & ORDERING
INFORMATION'. If Microtiter combs have been used, wells can be loaded by a multi channel pi-
pette. Wells that have been cast with microtiter combs of 26 teeth or less can be loaded 'directly',
i.e. the pipette tips fit sequentially into the gel wells.
NOTE: It is wise to always run a sample lane of a known 'standard ladder' to determine concentra-
tion and size of separated fragments after the gel run, and to aid in photo documentation and anal-
ysis.
5. Carefully slide the lid with attached power cords onto the unit. This will connect the power cords to
the banana plugs to complete the circuit. Plug the other end of the cords (4 mm, male) into an ap-
propriate power supply.
Take care to the proper orientation of the electrical field. Remember that nucleic acids are negatively
charged in an alkaline to neutral surrounding and therefore will migrate to the positively charged
anode. In general, the color coding for positively charged electrodes is red.
6. Turn on the power supply and run the gel at the appropriate voltage/current (see 'Technical proper-
ties'). You can observe the progress of electrophoresis by the visual migration of the loading dye.
Note that in 0.5 x TBE gels bromophenol blue co-migrates at 300 bp and xylene cyanol at 4 kbp
with the DNA fragments.
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Horizontal Mini Gel Systems
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