DNA Sequencing User's Guide
Overview of Sanger sequencing by capillary electrophoresis
During sample preparation, the DNA fragments in a sample are chemically labeled with fluorescent dyes. Using PCR, unlabeled deoxynucleotides (dNTPs) and dye-labeled dideoxynucleotides (ddATP, ddGTP, ddCTP, and ddTTP) are incorporated by Taq DNA polymerase into the growing DNA strands. The dye-labeled DNA fragments are separated by electrophoresis within the 96-capillary array of the ABI (Applied Biosystems) 3730xl DNA Analyzer. Once the fragments enter the detection cell, they pass through a laser beam. The light excites the attached dye labels causing them to fluoresce. A computer analyzes the fluorescence to determine the order of DNA bases in the strand.
Three files are created for each sequence:
- .seq — a simple sequence text file
- .ab1 — an electropherogram (sequence data plotted as a graph of relative dye concentration against time, plotted for each dye)
- .phd.1 — (Phred file) simple text file showing bases with quality values for each base
Sample Preparation
Culture
- ABI recommends HB101 or DH5a.
- Mv1190 and XL1Blue give variable results.
- JM101 usually doesn’t work.
Purification
Recommended by the Sequencing Core: Use a Qiagen kit or standard alkaline lysis miniprep purification followed by 13% PEG8000 precipitation. Resuspend DNA in dH2O.
What to avoid:
- Do not use Promega Magic preps.
- Avoid purifying DNA with phenol since residual phenol interferes with the sequencing reaction.
- Do not resuspend DNA in TE.
In general, whatever purification process is easiest to prepare should be fine. However, the cleaner the template provided, the cleaner and better the sequence read and read length returned. You are responsible for the purity of your samples...do NOT send contaminated samples!
Primers
Longer primers work better. T7, T3, and M13 forward and reverse primers are recommended. SP6 is less reliable. Custom primers should be 21- or 22-mers.
Preparing samples for PCR
Mix reagents in a small, thin-walled, 0.2 μl PCR tube.
Use the following recipe for the CLEAR pre-mix (or Dye Terminator Mix) reaction.
| Reagent | Quantity | |
|---|---|---|
| Template | ss DNA | 0.05–0.10 μg |
| ds DNA | ~0.50 μg | |
| PCR product | 100–200 bp | 1–3 ng |
| 200–500 bp | 3–10 ng | |
| 500–1000 bp | 5–20 ng | |
| 1000–2000 bp | 10–40 ng | |
| >2000 bp | 40–100 ng | |
| Primer | 3.2–6.4 pmol | |
| dH₂O | quantity sufficient | |
| Final volume | 9 μl |
Labeling the PCR tube
- Label each sample in the service request with your initials and a unique number (e.g., PIB1, PIB2, PIB3).
- Write above the line on the tube AND only write horizontally.
- Writing anywhere else, including the lid, will be rubbed off in the PCR machine, forcing you to repeat your sample.
- Do not include your primer or template on the label.
The Sequencing Core will add Dye Terminator Mix before running your samples.
SPECIAL NOTE about primer volumes
For Double-Stranded Plasmid DNA, increasing the amount of primer more than 6.4 pmol generally doesn’t cause problems. But don't add too much!
For PCR products, however, it is generally better to stay around 3.2 pmol or only slightly higher if the PCR product is very large.
Analyzing Data
Viewing the sequencing text file
Any simple text editor, such as TextEdit (Mac) or Notepad (PC), can open the .seq file. SnapGene Viewer and ApE are also options.
NOTE: If there is no .seq file and the .ab1 electropherogram file is 72 KB, your sample failed to give a readable sequence.
Analyzing the electropherograms
Several free programs are available to view the *.ab1 files and analyze your data.