Troubleshooting

No Data (No Signal) result is given when the signal strength of the fluorescent termination products is too low for the computer software to call bases. Below are common reasons why this occurs.

Reasons for "no data”	Insufficient amount of DNA template (see chart regarding DNA concentrations)
	Poor quality of DNA template (see Contaminants affecting DNA sequencing)
	Insufficient primer concentration (or no primer added)
	Primer Tm is less than 50 °C, the annealing temperature of the sequencing reaction is 50 °C
	Template does not have primer site--make sure that the correct primer is chosen for the vector being used.
	Primer does not anneal well to priming site. A set of primers may produce a PCR product, but one primer may not anneal as efficiently and therefore not work well for DNA sequencing, which is linear amplification, unlike PCR which is exponential amplification.

Short Read

Top-heavy data	Excess DNA template or primer or salt in the sequencing reaction results in top-heavy data because balance of the sequencing reaction is shifted towards generation of the shorter products. DNA purification using QIAEXII kit usually results in top-heavy data.
Gradual decrease in the signal	Repetitive DNA region
Abrupt decrease in the signal	Region of secondary structure or template sequence idiosyncrasies
	Poor quantification of primer and/or template, leading to top-heavy data

Multiple Peaks in the Sequence

PCR products	At the beginning of the sequence – primer-dimer sequence.
	All the way – PCR product was not purified from primers.
	Frame shift mutation
Plasmid DNA	After some good sequence – more than 2 colonies picked
	Slippage after homopolymer region in template.
Noisy sequence	The signal is too low (see “no data“)
	Signal is too high causing detector saturation, as result the software can’t call bases properly.
	Contaminated template (see Contaminants affecting DNA sequencing)
	Multiple priming sites or multiple primers
	N-1 signal – sequencing primer contaminated with not full length primer
"Spikes" in the sequence	Degraded DNA. Nuclease contamination and repeated freeze-thaw cycles can result in degradation of DNA. Spike location in these samples will not change the position after re-run.
	Contaminated template (see Contaminants affecting DNA sequencing)
	Problem with the polymer on 3730-Genetic analyzer. Contact Mobix lab within 24 hour from receiving this kind of result and we will repeat run free of charge.

Contaminants Affecting DNA sequencing

The sequencing reaction will fail if there is more than:

	A)	1mg RNA - 1ml E.coli culture gives 1 to 5 mg plasmid DNA but 100 to 500 mg RNA!
	B)	0.3% PEG - that’s 2.5ml left from a 100ml precipitation
	C)	0.5mM NaAc - that’s 0.5ml left from a 100ml precipitation
	D)	1.25% EtOH - that’s about 2ml left from a 100ml precipitation
	E)	0% phenol - in other words not even a trace!
	F)	0% chloroform - likewise!
	G)	5mM CsCl - that is also not very much from a CsCl gradient
	H)	5mM EDTA - dissolve your DNA in water or 10mM Tris pH8.5

If you RNase treat you must phenol extract and isopropanol precipitate to remove the digested RNA. Pipette off as much ethanol as possible from DNA pellets. Always wash DNA pellets with 70% ethanol/H2O, especially after phenol or chloroform extraction. Dry pellets before dissolving, and don’t use TE unless necessary.

Bio-Bars