DNA Sequencing
The Nucleic Acid lab uses two 48-capillary Applied Biosystems 3730 DNA Analyzers and all the necessary accessory equipment for DNA sequencing. A modified Sanger dideoxy chemistry, the ABI Prism BigDye Terminator Cycle Sequencing Kit version 3.1 is the method of choice. Data collection software v3.0 and sequencing analysis software v5.2 are used on both machines.
If samples are submitted before 9am the results will be returned the next morning. Researchers initiate the process by submitting a specific amount of template combined with the primer and a DNA Sequencing Request Form denoting: type of reaction to be performed, primer to be used in the reaction and the name of the template. The information is used to create plate maps for sequencing extension reactions, sequencing data collection, and sequencing analysis. The information is also used for tracking and billing purposes. Post sequencing, the electropherograms are manually analyzed for overall quality.
Short Reference for publications
The Nucleic Acid Shared Resource uses the BigDye Terminator Reaction Chemistry v3.1 for sequence analysis on Applied Biosystems 3730 DNA Analyzers.
Template DNA Amounts for PCR Products
| 100bp | 1ng | 1100bp | 55ng |
| 200bp | 5ng | 1200bp | 60ng |
| 300bp | 15ng | 1300bp | 65ng |
| 400bp | 20ng | 1400bp | 70ng |
| 500bp | 25ng | 1500bp | 75ng |
| 600bp | 30ng | 1600bp | 80ng |
| 700bp | 35ng | 1700bp | 85ng |
| 800bp | 40ng | 1800bp | 90ng |
| 900bp | 45ng | 1900bp | 95ng |
| 1000bp | 50ng | 2000bp | 100ng |
Plasmid: 450ng
Cosmid/BAC DNA: 900 ng template + 6.4 pmol primer in a total volume of 12 ul!
Primer amount: 6.4 pmol
Mix in a labeled 0.2 ml PCR tube in a total volume of 12 ul.
Primer Design for Taq Dye Deoxy Terminator Cycle Sequencing Reactions:
- Cost: The shorter the cheaper.
- Primers should generally be between 18-24 bases long to ensure good hybridization. Longer and shorter oligos seem to have a decrease in specificity.
- Aim for an annealing temperature of 60°C. Optimize GC content and length with the following formula: Tm=0.41(%GC)+69.3-650/L (L=length of primer).
- Primers should have one or more G- or C- residues at the 3’ end.
- Primers that have secondary structures (inverted repeats), or that can hybridize to form dimers, should be avoided.
- Homopolymeric regions (i.e. more than three or four, especially G or C) should generally be avoided.
Conversion from pmol to ng: pmol/µl = µg/ml x 1000 / MW
The safe way to determine concentrations is to use a spec!
The Nucleic Acid Shared Resource provides support for measuring DNA concentrations using a NanoDrop for single-tube assays and a Beckman DTX880 Multimode Plate Reader for 96-well plate assays.
Extinction coefficients for nucleic acids (NanoDrop):
Double-stranded (ds) DNA: 1 ODU ≈ 50 ng/ul
RNA: 1 ODU ≈ 40 ng/ul
Single stranded (ss) DNA, cDNA or oligos: 1 ODU ≈ 33 ng/ul
Betaine:
In some cases where the regular chemistry fails, e.g. GC-rich areas, repeats, bisulfite-treated samples, or 2° structures, we can achieve good sequencing data by adding betaine to the reaction mix.
We add 2 ul BigDye undiluted and 2 ul betaine undiluted from the 5 M stock to 6 ul of submitted template and primer.
The correct concentrations of template and primer are crucial.