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Preparing and Preparing and Loading Experimental Samples
Loading Samples Note Often you will need to dilute the PCR product from 1:5–1:20 in distilled, deionized H O
2
before loading 1 µL into the sample tube.
Step
1
Action
Prepare Loading Mix sufficient for loading 100 samples:
♦
250 µL deionized formamide
♦
50 µL GeneScan size standard (GeneScan-350, GeneScan-400 HD, or
GeneScan-500 recommended)
♦
25 µL Loading Buffer (25 mM EDTA, 50 mg/mL blue dextran)
! WARNING ! CHEMICAL HAZARD. Formamide is a known teratogen. It
can cause birth defects. Wash thoroughly after handling formamide. Obtain a
copy of the MSDS from the manufacturer. Wear appropriate protective
eyewear, clothing, and gloves. Wash thoroughly after handling formamide.
Note
2
Vortex gently for 3–5 seconds. Loading Mix lasts for 2 weeks at 2–6 °C.
To each sample tube add the following:
♦
3.0 µL Loading Mix
♦
1 µL PCR product
3
Place the samples in a heat block or thermal cycler for 5 minutes at 95 °C.
4
Remove the tubes from the heat source and place immediately in an ice-water bath.
5
Load 4 µL of sample into each gel lane.
Preparing the Matrix Samples
Step
1
Action
To each of four sample tubes add the following:
♦
2.5 µL deionized formamide
♦
2.5 µL of one of the matrix standards (Dye Primer, Fluorescent Amidite, or
NED matrix standard).
! WARNING ! CHEMICAL HAZARD. Formamide is a known teratogen. It
can cause birth defects. Wash thoroughly after handling formamide. Obtain a
copy of the MSDS from the manufacturer. Wear appropriate protective
eyewear, clothing, and gloves. Wash thoroughly after handling formamide.
2
Denature the samples for 5 minutes at 95 °C.
3
Cool the samples by placing directly on ice.
4
Run 4 µL of each of these four samples, either alone on a gel or in the first lanes of
an experimental run.
Note
To ensure the best possible quality matrix, load the matrix samples into
every other lane and leave at least one empty lane between the matrix samples and
the experimental samples.
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Microsatellite Analysis 8-15