DNA

Common Phenotypes and Solutions - DNA

Additional Sample or Ladder Peaks -- More peaks, often shorter, than expected

Most Probable Causes

• Chip contaminated -- Wear powder-free gloves
• Dust particles in separation channels -- Clean the electrodes; load the chip immediately after taking it out of its sealed bag

Probable Causes

• Sample degraded or contaminated -- Always wear gloves when handling chips and samples to prevent them from getting contaminated
• Chip preparation with cold reagents -- Prepare a new chip allowing reagents to warm to room temperature for 30 minutes before use

• Chip not properly primed -- Use a new chip; check chip priming station using the seal test; check if clip and base plate of priming

  station are in correct position

• Vibration of bioanalyzer -- Don't touch the bioanalyzer during a run; remove vibration devices from bench

Least Probable Causes

• DNA ladder degraded -- Check expiration date of reagents

Spikes -- Large narrow peaks

Most Probable Causes

• Vibration of bioanalyzer -- Don't touch the bioanalyzer during a run; remove vibration devices from the bench

• Chip/gel-dye mix contaminated with particles -- Prepare new chip with new gel-dye mix; wear powder-free gloves only; don't touch the

  underside of the chip; don't touch the wells of the chip; clean the electrodes; load the chip immediately after taking it out of its sealed bag

• Dye agglomerates present in the gel-dye mix -- Use dye concentration according to the Reagent Kit Guide

Probable Causes

• Chip preparation with cold reagents -- Prepare a new chip and allow reagents to warm to room temperature for 30 minutes before use

Least Probable Causes

• Power outlett -- Install power filter

Low Sensitiviy -- Many smaller peaks

Most Probable Causes

• Dye concentration too low -- Use dye concentration according to the Reagent Kit Guide
• Pipetting error during preparation of mixtures -- Check dilution procedure and calibration of pipette
• Chip pipetting error -- Pipette new chip; always insert the pipette tip to the bottom of the well when dispensing the liquid and holding the pipette at a slight angle will ensure proper dispensing of the liquid; use appropriate pipette tips

Probable Causes

• Fingerprint on focusing lens or on the backside of the chip -- Clean lens; do not touch the underside of the chip
• Insufficient vortexing of chip -- Vortex chip for 1 minute only using the IKA shaker for chip vortexing and adjust to proper speed

Least Probable Causes

• Chip contaminated -- Wear proper gloves only; don't touch the underside of the chip or the wells; clean the electrodes; load the

  chip immediately after taking it out of its sealed bag

• Vibration of the bioanalyzer -- Don't touch the bioanalyzer during a run; remove vibration devices from bench
• Autofocus failure -- Check autofocus using the Diagnostics test

Missing Peaks -- No or too few peaks observed; marker peak may be absent

Most Probable Causes

• Too high salt concentration in sample -- Refer to the maximum sample buffer salt concentration as specified in the reagent kit

  guide; dilute samples with deionized DNase-free water if necessary

• Clogged gasket and plastic adapter of priming station -- Check chip priming station using seal test; clean/replace gasket and

  plastic adapter if necessary

• Wrong settings of the chip priming station -- Check if clip and base plate of priming station are in the right position
• Current leaks due to contaminated electrodes -- Clean electrodes with analysis-grade water and a toothbrush

Probable Causes

• Laser broken -- Perform Laser/LED/Optics test and Autofocus test

Least Probable Causes

• Autofocus failure or high voltage power supply defective -- Check autofocus and high voltage power supply by Diagnostic test
• Sample wells filled with water -- Refer to the Reagent Kit Guide for proper preparation of the chip

Missing Upper Marker

Most Probable Causes

• Alignment of upper marker not working properly -- Manually set the upper marker: in the electropherogram tab, highlight the

  appropriate peak in the peak table; with a right-mouse click, select "Manually Set Upper Marker"

• Upper marker digested by restriction enzymes -- Inactivate restriction enzymes by adding EDTA or heat inactivate according to

  manufacturers instructitons

Probable Causes

• Too high salt concentration in sample -- Refer to the maximum sample buffer salt concentration as specified in the reagent kit

  guide; dilute samples with deionized DNase-free water if necessary

Broad Peaks

Most Probable Causes

• Current leaks due to contaminated electrodes -- Clean electrodes with analysis-grade water and a toothbrush

• Clogged gasket and plastic adapter of priming station -- Check the priming station using the seal test; clean/replace gasket and

  plastic adapter if necessary

• Wrong settings of the chip priming station -- Check if clip and base plate of priming station are in the right position
• Dye concentration too high -- Use dye concentration according to the Reagent Kit Guide

Probable Causes

• Genomic DNA or cDNA contamination -- Check DNA preparation procedure

Baseline Dips -- A wider dip in the baseline than a spike

Most Probable Causes

• Too high sample concentration -- Use sample concentration according to the DNA Reagent Kit Guide
• Sample impurities: e.g. gDNA, ssDNA, etc -- Check DNA-isolation protocol; if possible, clean up samples

Probable Causes

• Dye concentration too low -- Use dye concentration according to the Reagent Kit Guide

Least Probable Causes

• Autofocus failure -- Check autofocus using the autofocus test in Diagnostics

Baseline Noise -- Baseline is made up of many small, sharp up and down peaks

Most Probable Causes

• Fingerprint on focusing lens or on the backside of the chip -- Clean lens; do not touch the underside of the chip

Probable Causes

• Autofocus failure or high voltage power supply defective -- Check autofocus and high voltage power supply under Diagnostics

Least Probable Causes

• Chip contaminated -- Wear powder-free gloves only; don't touch the underside of the chip or the wells; clean the electrodes; load

  the chip immediately after taking it out of its sealed bag

Baseline Jumps -- Baseline does not remain uniform and jumps up or down very suddenly

Most Probable Causes

• Vibration of the bioanalyzer -- Remove vibration devices, such as vortexers and vacuum pumps, from the bench
• Instrument lid was touched during the run -- Don't touch the bioanalyzer during a run

Least Probable Causes

• Laser defective -- Check Laser by using the Diagnostics test

Wavy Baseline -- Baseline not uniform across x-axis

Most Probable Causes

• Leak current due to dirty electrodes -- Clean electrodes
• Detergents in PCR-buffer lowers surface tension in wells of chip, which leads to liquid spill on the top of the chip during vortexing -- Prepare a new chip and lower the vortexing speed or mix samples manually
• Dye concentration too low -- Use dye concentration according to the Reagent Kit Guide

Probable Causes

• Wrong settings of the chip priming station -- Check if clip and base plate of priming station are in the right position
• Clogged gasket and plastic adapter of priming station -- Check the priming station using the seal test; clean/replace gasket and plastic adapter if necessary

Least Probable Causes

• Changes of ambient temperature of more than 5C during the run -- Place bioanalyzer in thermally stable environment
• High voltage power supply defective -- Check high voltage power supply using the Diagnostics test

Late Migration -- Peaks do not appear until late

Most Probable Causes

• Vortex speed too high -- Vortex at lower (medium) speed; for chips, use only the IKA vortexer
• Leak current due to dirty electrodes -- Clean electrodes
• Detergents in PCR-buffer lowers surface tension in wells of chip leading to liquid spills on top of the chip during vortexing -- Prepare a new chip and lower the vortexing speed or mix samples manually
• Dye concentration too low -- Use dye concentration according to the Reagent Kit Guide

Probable Causes

• Vortex adapter not connected tightly -- Press vortex adapter tightly on mount; replace vortex adapter
• Clogged gasket and plastic adapter of priming station -- Check chip priming station using the seal test; clean/replace gasket and plastic adapter if necessary
• Wrong settings of the chip priming station -- Checkif clip and base plate of priming station are in the right position
• Genomic DNA or high molecular weight DNA contamination -- Check DNA isolation protocol

Least Probable Causes

• Changes of ambient temperature of more than 5C during the run -- Place bioanalyzer in thermally stable environment

Peak Tailing -- After expected peak, second peak observed very close to expected

Most Probable Causes

• Restriction enzymes may cause peak tailing -- Use different restriction enzyme; appy additional purification step

return to HomePage