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Production of the Model 4300 has been discontinued as of January 1, 2014.
Service and support may become limited. Please contact your local sales office.

Troubleshooting

Troubleshooting AFLP® reaction

Problem Possible Cause Possible Solution
No bands or weak bands IRDye® infrared dye-labeled TaqI primer(s) not added or degraded. Be sure to add EcoRI primer(s) and avoid exposure to labeled primers to light.
Not enough template DNA. Be sure to have ≥ 75 ng of DNA per restriction digest reaction.
DNA contaminated (e.g., high salt, EDTA, SDS, or protein). Extract with phenol/chloroform followed by ethanol precipitation.
Incorrect PCR conditions. Verify the cycling program temperature, cycle number, and time; make sure that the thermal cycler is operating correctly.
Evaporation during thermal cycling. Cover the reactions with mineral oil or liquid wax; centrifuge briefly before incubation; check that caps fit correctly when using thermal cyclers with heated lids.
Too few high molecular weight bands Sub-optimal primer pair. Some primer combinations are not ideal for some species. Use suggested primer pair for a given species.
Sub-optimal quantities of primer, pre-amplified DNA, or both. Try a different quantity of IRDye EcoRI primer (0.3 - 0.8 μL per 11 μL selective PCR reaction) and a different dilution of pre-amplified DNA template (1:10 - 1:50).
Many bands of high molecular weight Partial digestion. Purify DNA.
Bands only part of the way up the gel Poor DNA, low polymerase activity, or both. Purify DNA templates and use fresh Taq DNA polymerase.
Missing lanes (non-specific, variable) Evaporation during thermal cycling. Cover the reactions with mineral oil or liquid wax; centrifuge briefly before incubation; check that caps fit correctly when using thermal cyclers with heated lids.
Pipetting error. Verify addition and mixing of all reaction components.
Sub-optimal duplexed AFLP results Competition effects of two labeled EcoRI primers over one MselI primer. Choose two labeled EcoRI primers with similar predicted Tm. If the problem persists, try monoplex PCR first and pool samples later.

Troubleshooting AFLP® reaction

Problem Possible Cause Possible Solution
Blurry bands Improper gel formation. Recast gel using fresh solutions and allow gel to polymerize ≥ 45 minutes.
Smeared Bands Too much labeled primer(s). Try less labeled EcoRI primer(s).
Samples not denatured. Add 5 μL of stop/loading buffer and heat the sample at 95 °C for 3 minutes immediately before loading gel.
Incorrect electrophoresis conditions. For running a 25 cm gel (LI-COR® 6.5% KBPlus, P/N 827-05607), set temperature at 45 °C, voltage to 1500-2000 volts, current to 40 mA, power to 40 W, and scan speed to 2 (Model 4300).
Differences between gel and running buffer. Verify that the buffer in the gel and the running buffer are the same concentration (1X TBE).
Wavy Bands Gel surface did not polymerize evenly. Recast a gel and make sure the wells are free of excessive urea and unpolymerized gel solution.
Wells not formed properly. Make sure to pull a rectangular-tooth comb straight out carefully.
Binding silane not used in gel preparation. Apply binding silane to front plate before casting gel.
Air bubbles in gel. Recast gel, tap gel apparatus to ensure smooth flow of gel.
Smiling Bands Uneven gel thickness. Do not over-tighten the rails or upper buffer tank to avoid uneven thickness of the gel.
Outer lane(s) missing Comb not centered. Be sure to insert the comb in the center of the gel.
Improper running buffer. Be sure to use freshly made buffer (1X TBE) to perform gel electrophoresis.


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