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PippinHT Operations Manual
11-2
460005 Rev G
11.2 Sample loading procedure
1.
Re-check the buffer level in the sample wells.
Make sure that sample wells are
completely full to the top with electrophoresis buffer. Top off with additional buffer, if
necessary.
2.
Remove 3
0μl of
buffer the samples wells.
Take care not pierce the agarose with the
pipette tip(s). There is gel on all sides and bottom of the sample well. In addition, there is
an agarose “chimney” surrounding the top of the sample well that protrudes up through
the cassette cover (see
Figure 11.1
). When removing buffer, some users find it useful to
immerse the pipette tip just below the surface of the buffer and follow the liquid level
down with the tip as the buffer is removed. When buffer removal is completed, there will
be ~10ul of buffer left in the well.
3.
Load 25
μl of sample to the sample wells.
When adding sample, place tip of pipette just
below the surface of the buffer, and follow the liquid level up with the tip as the well fills.
Don’t be concerned if the sample well slightly overfills. The density of the sample will
allow it to sink before it can flow out of the well.
11.3 Loading less than 12 samples, and reusing a gel cassette
It is possible to run less than 12 samples on a PippinHT gel cassette. When a size
selection protocol is programmed, only the lane-pairs which have been assigned a
function (size selection or external marker) will be used. Any remaining lane-pairs can
be resealed (with PCR tape, such as MicroSeal A or similar) and reused at a later time
up to the expiration date of the cassette.
There are some very important considerations when running a partial gel cassette:
•
Users should double-check that the size selection protocol matches the lane-pairs
that will be loaded.
•
Users should double-check that the unused lanes are de-selected in the size
selection protocol (
Figure 11.2
)
•
Tightly reseal the unused lane ports with PCR tape after the initial run
•
Clearly mark the unused lanes with a lab marker
Figure 11.2.
A de-selected lane-pair. The programming mode
and “Ref Lane” field must be OFF.
