4 prepare the samples and the dna ladder, 5 prime the chip – Bio-Rad Experion DNA Analysis Kits User Manual
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3.4 Prepare the Samples and the DNA Ladder
Each chip can analyze up to 11 DNA samples. All wells of a chip must be filled for the
electrophoresis station to operate properly.
1. Remove the DNA ladder from storage, vortex to mix, then centrifuge briefly. Place the ladder on ice.
2. Prepare the samples as necessary to meet the following requirements for analysis:
n
DNA concentration — Samples should have a DNA concentration within the linear range
of the assay (0.1–50 ng/μl). Make an appropriate dilution of the sample, for example in
TE buffer (10 mM Tris, 1 mM EDTA, pH 8), as needed
n
Salt concentration — The concentration of salt in the DNA sample must be <250 mM KCl
or NaCl, 15 mM MgCl
2
. High salt concentrations increase conductivity, reducing the
amount of sample injected onto the chip and affecting assay sensitivity and potentially
reducing quantitation accuracy and precision. Make an appropriate dilution of the sample
in TE buffer or water, as needed
n
Restriction enzyme deactivation — Restriction enzyme digests may require the addition
of EDTA to chelate metal ions and/or heat inactivation of the enzyme prior to running the
chip. Failure to inactivate the restriction enzyme may result in degradation of the internal
DNA markers, inaccurate sizing, or peak broadening
3.5 Prime the Chip
1. Pipet 9 µl GS into the highlighted well labeled GS, gel priming well (Figure 3.1). Insert the pipet tip
vertically and to the bottom of the well when dispensing. Dispense slowly to the first stop on the
pipet, and do not expel air at the end of the pipetting step.
2. On the priming station, set the pressure setting to C and the time setting to 3 or 1, as specified by
the alphanumeric code on the chip (Figure 3.1).
Fig . 3 .1 . Experion DNA chip . The locations of the gel priming well
(GS, highlighted) and alphanumeric priming codes are indicated.
Gel priming well
Priming codes
Start the run within 5 min of priming and loading the chip. For help with chip loading, refer to the
Experion DNA 1K and DNA 12K Analysis Kits