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Table 11-6 Problems with Current Observation Possible Causes Recommended Actions No current Incorrect TBE buffer formulation Use correct TBE buffer concentrations: Instrument set up incorrectly Broken instrument parts Corrupted firmware ♦ 10X TBE in the gel (for a final concentration of 1X) ♦ 1X TBE running buffer Verify that: ♦ The electrode leads are secure and plugged in. ♦ The bottom and top of the gel are immersed in running buffer. Check for breaks in: ♦ Electrode leads ♦ Buffer chambers ♦ Platinum wire in buffer chamber Resend firmware by performing a total reset. Table 11-7 Problems with Signal Strength and Quality Observation Possible Causes Recommended Actions Signal too low, i.e., peak heights are lower than usual Insufficient sample added to some or all lanes Did you add a full 1.5 µL of PCR product to formamide/size standard mix? If not, add 1.5 µL PCR product to formamide/size standard mix. If so, concentrate your PCR product before adding to formamide/size standard mix or examine the efficiency of the PCR. Check pipette calibration. Cassette not flush with back heat transfer plate and alignment pins (ABI PRISM ® 377 DNA Sequencer only) Place cassette flush against back heat transfer plate. Difficulty in loading sample because wells not flushed Flush the wells prior to loading the gel. IMPORTANT The spacers must touch the alignment pins. You should be able to see the alignment pins pressing against the spacers. Troubleshooting 11-9
