Failed Samples

1. Primer has no annealing site.
2. Insufficient template.
3. Contaminated template.
4. Poor priming design, e.g. low melting temperature.

Noise

1. Low signal strength.
2. High signal strength (saturating the detector).
3. Contaminated template.
4. Multiple templates in sequencing reaction.
5. N-1 primer.
6. Frame shift mutation.
7. Primer-dimer contamination in PCR sequencing.
8. Slippage after homopolymer region in template.

Gradual and abrupt signal losses

1. Region of secondary structure in the template (see note#2 below).
2. Template sequence idiosyncrasies.
3. Poor quantitation of primer and/or template, leading to top heavy data.

Poor quality/quantity template (see note#1 below).

1. Residual salt or organic chemicals carried over from the template preparation.
2. Incomplete removal of cellular components such as RNA, proteins, polysaccharides, and contaminating chromosomal DNA.
3. Degradation of DNA during storage.
4. More than one template DNA in the sequencing reaction.

EDTA and salt concentration
High EDTA and salt concentration will decrease the signal strength/ readlength.

Primer problems
Poor priming resulting in weak or no signal.

1. Melting temperature is too low due to low GC content, short primer length.
2. Secondary structure of the primer, particularly at the 3' end
3. Secondary structure of the template in the region of hybridization.
4. Incorrect primer concentration.
5. Priming site not present.

1. Secondary hybridization site, which results in many extra peaks.
2. Impure primer. You may see a shadow of N-1.

Note #1; Poor quality/quantity template
The directions for cell growth shall be followed (cautious use of Terrificbroth). Overloading the Qiagen resin yields poor quality DNA.

RNA competes with plasmid DNA binding to the Qiagen resin. It also affects the quantification of the DNA if this is done spectrophotometrically. The presence of RNA contaminants can be determined by analysis of the template preparation on agarose gels.

When estimating the amount of DNA on an agarose gel, be sure to load a low amount of DNA (i.e. 30ng-50ng per lane) with a marker of known concentration (not degraded itself). Low film exposures (˜1-2 sec) and an OD (260) is also advised. Overloaded gels and longer film exposures will likely give poor estimations of amount.

Spectrophotometer: the A (260/280) ratio should be 1.7-1.9. Smaller ratios usually indicate contamination by protein or organic chemicals. RNA contamination will affect DNA quantitation greatly. Absorbance measurements of highly concentrated (O.D. > 1.0), very dilute (O.D. > 0.05) DNA sample, and PCR products can be inaccurate.

One A (260) unit of single stranded DNA contains 33 ng/ul.
One A (260) unit of double stranded DNA contains 50 ng/ul.

Neither gel or OD show the presence of contaminating salts, residual ethanol or EDTA contamination, which interfere with Taq polymerase activity for sequencing.

Salts can be removed with spin columns such as Centri-sep (Princeton Separations) before sequencing. The isopropanol-precipitated DNA shall be washed with 70% ethanol to remove excess salt. Wash the DNA pellet at least once as directed with 70% ethanol.

The template DNA shall be dried completely before final resuspension in H2O. To remove residual ethanol, dry the DNA for 5 min. in a properly operating speedvac. If air-drying, make sure that the DNA is dry (no fluid in the tube, the DNA pellet does not look wet). When air drying, a brief 15 min incubation of the open tube at 65°C is often sufficient to completely dry.

Note #2: Difficult sequencing regions may result in false reading of the actual sequence. An example of a difficult sequencing region is a homopolymer of A, T, C, or G bases in the template. Homopolymer regions typically cause drops in signal intensity, noisy data, and/or enzyme slippage. GC repeats produce abruptly stopped sequencing data. Any ambiguous data proceeding a difficult sequencing region may reasonably be obtained by sequencing the complimentary strand. Sequencing with dGTP Big-Dye Terminator shall be helpful in some cases.