GO TO: The DNA Sequencing Group The Protein Sequencing Group The Peptide/DNA Synthesis Group The Microchemistry Group The Amino Acid Analysis Group The Electrophoresis Group The Mass Spectrometry Group

Problems associated with Templates		Problems associated with Primer
Poor Template Quality Impurities such as protein, RNA and salts in template DNA will produce high background, and poor quality data. Additionally, some peaks will have fairly large signal underneath the correct signal. Low Template Concentration Low signals, noisy data, errors and ambiguities are high. High Template Concentration Peaks in sequencing data show trailing and may be accompanied by a weaker signal. Template Composition G/C rich template (particularly when % GC exceeds 62%) This template is more difficult to sequence. The addition of DMSO helps the denature them to increase it read through and resolution. A/T rich template Some additional G or C noise may be observed throughout. Similarly, A and T noise may appear in G/C rich template.		Poor priming resulting in very weak signal Melting temperature too low: low G/C content or short Primer Secondary structure of primer, particularly at the 3' end Secondary structure of template in region of hybridization Primer Design Rules Sequence: Avoid 3 or more G or C at the 3' end Avoid a T at the 3' end Avoid complementary sequences within a primer and between primers Length 18-30 bps GC content 40-60% Tm Tm=2'C X (A+T) + 4'C X (C+G) Concentration 0.1-0.5uM

Contaminants		Noisy Data
Amounts of Contaminants Tolerated in Sequencing Reaction Type of Contaminant Concentration RNA 1ug PEG 0.3% NaOAc 0.5mM Ethanol 1.25% Phenol/Chloroform 0% (they denature dyedeoxy terminators) CsCl 5mM EDTA 0.25mM		Noisy Data:  Overall weak signal resulting in noisy data.  Presumably this is related to the tighter duplex and greater difficulty of denaturing the template and keeping it denatured, as well as the presence of secondary structure. Reasons for Noisy Data 1.  Unbalanced template base composition results in high baseline noise in one or more colors. 2.  Secondary hybridization site will generate many peaks over peaks. More stringent annealing conditions can eliminate this 3.  Impure primer; may see a "shadow sequence" 4.  Incorrect primer sequence (for custom primers) 5.  Mismatch on 3' end will generally result in completely flat line or no signal.

Authored by Jeremy Medalle