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Troubleshooting PCR Product Detection Topics This section offers troubleshooting suggestions for the following problem areas: ♦ Problems with gel or gel image (page 11-7) ♦ Problems with automatic data analysis (page 11-8) ♦ Problems with current (page 11-9) ♦ Problems with signal strength and quality (page 11-9) ♦ Problems with peak number and position (page 11-11) ♦ Problems with peak quality and resolution (page 11-13) Table 11-4 Problems with Gel or Gel Image Observation Possible Causes Recommended Actions Misshapen wells Suction when comb removed When removing the comb: Note If only a few wells are misshapen, you can still load the gel using only those lanes with flat, well-formed wells Wet comb or well-former 1 Lay gel flat. 2 Pour 1X TBE over comb. 3 Remove comb slowly. Ensure that comb is clean and dry before inserting into gel. Note To ease insertion of comb, you can “wet” the comb with acrylamide (or the appropriate gel polymer). Severely bowed gel image Air bubbles trapped when comb inserted Ensure that no air is trapped by comb. Clamping bottom of gel plates Do not clamp the bottom of the gel. Note Use clamps of equal tension along the side of the gel. The clamps must be placed over the spacers. Gel extruded between plates into upper buffer reservoir After cleaning plates, soak for 10 minutes in 3M HCl, then rinse with distilled, deionized H2O. Remake gel. Note This will remove any residual charge on the plates that might cause the gel to become oppositely charged. Red or green smearing on gel Gel dried out before running Before storing a poured gel, wrap the gel ends with damp Kimwipes and plastic wrap. IMPORTANT Use gels within 24 hours of pouring. Appearance of donut-shaped band (with black central hole) Too much sample loaded Load less sample or remake sample with a higher dilution. Large migrating region showing no fluorescence Plates not rinsed free of Alconox Rinse plates thoroughly with distilled, deionized H2O. Troubleshooting 11-7
