978-922-1832
●
www.sagescience.com
●
BluePippin Operations Manual 460013 Rev F
11-1
11
Loading Samples
Proper sample loading is critical for best performance of BluePippin cassettes. For
maximum reproducibility and accuracy, the sample should travel through the central
section of the gel column, and should be bounded on all four sides by uniformly
conductive media
– either gel or electrophoresis buffer. The goal of the loading
procedure is to produce this geometry in the sample loading well, as illustrated in the
bottom section of
Figure 11.1
. Properly prepared samples will be 40 ul in total volume
consisting of 30 ul of DNA mixed with 10 ul of BluePippin internal standard mix or
loading solution. The loading solution contains concentrated Ficoll as a densifying agent
(see the previous chapter on Sample Preparation for details), and therefore the samples
will sink and form a high density layer beneath the electrophoresis buffer when pipetted
slowly into the sample wells. If there is insufficient conductive buffer over the sample,
the electrophoretic forces lines will curve upward as the sample exits the well (see
Figure 11.1
, upper section), and the sample will be drawn to the top of the cassette
where it can travel out of the gel into the gap between the gel column and the plastic top
of the channel. Sample moving in this gap will travel at a different rate than the sample
inside the gel column, and will lead to elution of undesired size fractions in the eluted
material. In such cases, the contaminating DNA will usually (but not always) be higher in
molecular weight than the selected DNA.
Figure 11.1
. An illustration of the electrophoretic effect on a
DNA sample when a sample well is not completely filled.