Rebecca and John Moores UCSD Cancer Center DNA Sequencing Shared Resource

Purify Template
You must have a singular template product.
If you have a plasmid product, be sure to remove any bacterial genomic DNA.
If you have a PCR fragment product there are two options: Qiagen Qiaquick column purification or Exonuclease/SAP digestion.
Exo/SAP digestion is a simple method of removing excess unincorporated nucleotide triphosphates and primers. Exonuclease activity degrades the single stranded primers, while the phosphatase activity cleaves the gamma phosphate on the dNTPs, rendering them inert nucleosides. (USBweb.com has a kit for this called ExoSAP-IT, part number 7820001). If you are receiving nonspecific priming results for your PCR fragment sequencing data, please consider using this kit before sample submission.
Purification must remove all salts, EDTA, alcohols, protein, RNA, detergents (i.e. Triton X-100 and SDS), cesium, and phenol from your sample.
Your submitted sample should consist of only DNA and ddH2O. The presence of excess contaminants interferes with the sequencing reaction and electrokinetic injection of the sequencing products on our 3130XL Genetic Analyzers. This can result in weak signal strength, noisy data, or even reaction failure.
Achieve an O.D.260/280 between 1.8 and 2.0
This is important. If you submit templates that are out of this range, there is a good possibility that your reactions will fail or have very poor quality due to contamination and/or DNA concentration problems. Templates that are prepped together in one "batch" may have varying yields and levels of contamination, so measuring the concentration and purity of one template and assuming the rest will have the same concentration and purity is BAD.
Accurately Quantify Template
We operate ABI 3100 Genetic Analyzers which are capillary instruments that are very sensitive to DNA concentration. You are responsible for submitting your templates at the proper concentrations. Too much template will lead to shorter read length and/or noisy data. Insufficient template concentration may lead to weak signal strengths and noisy data or a failed sequencing reaction. We will not dilute templates that are submitted at concentrations higher than our recommended concentration. The best method of quantification is via fluorometer. If your template is contaminated, a spectrophotometer reading will be higher than the actual amount of DNA. Quantifying by eye on a gel is generally not accurate enough.

Target Template Concentrations

PCR products 50 ng/µL

Plasmid: 300 ng/µL

Cosmid 400 ng/µL

ssDNA 200 ng/µL

BAC DNA 400 ng/µL

DNA should be suspended in 6µL of ddH2O at the recommended concentration. For example: You are submitting a plasmid, and sequenced with one primer. 6 µL x 300 ng/µL = 1800 ng total DNA required for one reaction. Each µL should have 300 ng of plasmid DNA in it. Template must be at this correct concentration since DNA is not amplified as in regular PCR. If you do not have this much template, please call us to discuss your sample prep in more detail.
Visualize Template Quality
You have the option to verify your template's quality by running the template on Agarose Gel (or acrylamide) just prior to submission. Please make sure that it is run at an appropriate gel percentage, length of time, and voltage. Low voltage and longer running times provide better separation conditions. Also, TAE will not heat up the agarose gel as much as TBE. There should be one clearly defined band in the gel.
Determine sequencing Strategy
There are certain sequence motifs and secondary structure issues that may cause problems with sequencing. We will help you sort these problems out and suggest DNA sequencing strategies, but we can not guarantee that any particular sequencing strategy will solve difficult template problems and produce readable sequence.

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