BioVentures :: Technical Resources - Navigating the Universe of Genetic Diversity

Navigating the Universe of Genetic Diversity

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DMG Diversity Navigator™ Troubleshooting

Preparation of Sequencing Template
Cleanup of Sequencing Product
Sequencing and Analysis

Preparation of sequencing template

Observation	Possible Causes	Questions(?)	Recommended Actions
There was either weak PCR product or none at all.	An incorrect amount of one or more reagents was used or a reagent was omitted.	Were all reagents included and was the correct amount used?	Set up the reactions again using the correct amount of each reagent.
	Incorrect cycling conditions were used.	Has the thermal cycler program been checked?	If the program was incorrect, re-program the cycler and set up the reactions again.
		Were the recommended conditions followed?	Follow the recommended conditions.
		Was the initial denaturation set to the polymerase manufacturer’s recommendation?	Use the polymerase manufacturer’s recommended initial denaturation.
	The quality of the sample DNA was poor or an insufficient amount was used.	Was 10 ng of good quality sample DNA used? Does the sample DNA work with other primers? Did the control DNA amplify?	If the control DNA amplified well but other sample DNAs did not, clean the samples or increase the amount of sample DNA.
	No sample DNA was used.	Did the control DNA amplify?	If the control DNA did not amplify, the sample DNA may have been omitted. Set the reactions up again including the sample DNA.
	If the product was weak, conditions may have been such that more cycles are needed.	Were bands clean but weak?	Depending on the weakness of bands, put the plate back on the thermal cycler and try 5-10 cycles more than the recommended 35 cycles. Refer to BioVentures website Detailed Protocols/PCR From Genomic DNA.
	The enzyme concentration was too low.	Have you checked the manufacturer’s recommendation for enzyme concentration?	Set up the reactions again using the manufacturer’s recommended amount of enzyme.

Observation	Possible Causes	Questions(?)	Recommended Actions
Extraneous bands are seen in the PCR product.	The enzyme concentration was too high.	Have you checked the manufacturer’s recommendation for enzyme concentration?	Set up the reactions again using the recommended amount of enzyme.
	Incorrect cycling conditions were used.	Has the thermal cycler program been checked?	If the program was incorrect, re-program the cycler and set up the reactions again.
	The annealing temperature was too low	Was a temperature less than the optimal listed temperature used?	Set up the reactions again using the optimal listed temperature.
	The annealing temperature was too low	Was a temperature used that was between the optimal temperature and the lower limit of the listed range?	Increase the annealing temperature 2-3° and set up the reactions again.
	Too much or poor quality sample DNA may have been used.	Was 10 ng of good quality sample DNA used? Does the sample DNA work with other primers? Did the control DNA amplify?	If unsure, clean the sample DNA or use less of it.
	Too much or poor quality sample DNA may have been used.	Did the above recommended action fail to help?	Try nested PCR, if available, using 2 µl of a 1:1000 dilution of the product.
	If nested PCR was performed, too much PCR product was used.	Was 2 µl of a 1:1000 dilution of PCR product used per reaction?	Repeat PCR using 2 µl of a 1:1000 dilution of PCR product per reaction.

Observation	Possible Causes	Questions(?)	Recommended Actions
The size of the amplicon cannot be determined due to poor resolution of the marker bands.	The gel was not run long enough.	Is there enough space below the amplicon so that the gel can be run further?	If so, run the gel further.
	An inappropriate percentage gel was used.	What size was the amplicon? What percentage agarose was used?	Generally, use 4% agarose for bands up to 1000 bp; use 1-2% agarose for larger bands.
A DNA band is seen in the negative control.	The band may be spillover from the loading of adjacent wells on the gel.	Is the band relatively weak?	Run the negative control again on a mini-gel with an empty well on either side. Run a marker beside one of the empty wells.
A DNA band is seen in the negative control.	There is contamination in the negative control or one or more of the reagents.	Have you re-run the product on a gel to see if it is merely spillover? Have you seen evidence of contamination in other PCR products?	Carefully make up a new negative control and do a trial PCR. If that product is positive, it may be necessary to take steps to find and rid the lab of the contamination.
The 25% glycerol and sequencing template dry too slowly.	No heat or insufficient heat was used.	Was a SpeedVac® system or an incubator used?	The heat may be safely set at 70°C for either system.

Cleanup of Sequencing Product

Observation	Possible Causes	Questions(?)	Recommended Actions
Less than the expected volume comes through the Sephadex™ matrix.	The Sephadex™ matrix has dried out.	Is the matrix out of date? How was the matrix stored?	Re-wet the dry wells with distilled water and centrifuge again or make a fresh matrix.
More than the expected volume comes through the Sephadex™ matrix.	There was insufficient centrifugation prior to the addition of the sequencing product.	How long was the plate centrifuged prior to the addition of the sequencing product?	Dry this volume down in SpeedVac® or incubator and proceed.

Sequencing and Analysis

Observation	Possible Causes	Questions(?)	Recommended Actions
There was no signal or weak signal.	There was too little or poor quality sequencing template.	Does the PCR gel show a single clean band of sufficient yield?	Use more template if there was low yield; if the product was of poor quality, consult troubleshooting the preparation of sequencing template chart; more dye terminator may help.
There was no signal or weak signal.	An insufficient amount of primer was used.	Was the recommended amount of primer used?	Consult the supplied literature and use the recommended amount of primer.
There was high background.	Poor quality sequencing template could cause this.	Does the PCR gel show extraneous bands?	See the PCR troubleshooting chart.
Multiple DNAs appear in the chromatogram.	Poor quality sequencing template was used or more than one primer was used in the sequencing reaction.	Does the PCR gel show extraneous bands? Was more than one primer used in the sequencing reaction?	If the PCR gel shows poor quality product, consult troubleshooting the preparation of sequencing template chart; if more than one primer was used, set up the sequencing reactions again with one primer.
Excess dye terminator peaks interfere with results.	Sequence reactions were not cleaned appropriately.	Was the Sephadex™ matrix fresh?	If not, set up fresh sequence reactions.
Excess dye terminator peaks interfere with results.	Too much dye terminator was used.	Was the volume used within the recommended range?	Decrease the amount of dye terminator.