Both the DNA sample and the primer must be diluted in water and quantified either by fluorescence dye, agarose gel or OD 260nm (but only if no RNA is present). We require 5 µl of DNA and 5 µl of primer (1 pmol/µl = 1 µM) for EACH reaction. The Tm of the primer should be greater than 50 °C and the size should be 18 - 25 bases for optimum DNA sequencing. We have some common primers available for sequencing free of charge (please check list to see if the primer you require is on this list).

McMaster researches should drop off samples in our lab LSB-B123 Monday to Friday from 8:30 until 17:00.
External customers should send DNA samples and sequencing primers in 1.5 ml eppendorf tubes by overnight courier at room temperature. Tubes should be sealed with parafilm, and shipped in a padded envelope to the following address:

Mobixlab
McMaster University, LSB-B123
1280 Main St. W.
Hamilton, ON
L8S 4K1

Template Preparation

The DNA concentration must be quantified either by fluorescence dye, agarose gel against Mass Ruler, or OD 260nm, (but only if no RNA is present)(see Contaminant limits). Too much DNA will result in top-heavy data and shortened sequence read length. Too little DNA will result in no sequencing data

PCR PRODUCTS: Do not forget to remove primers! Qiagen column (or similar), or gel purification work well.
Check the DNA amount after purification by running 1 µl of DNA on an agarose gel - band should be visible.

PLASMID DNA: Qiagen column for small or large scale usually produces clean DNA, adding an extra wash step will give better results. Alkaline lysis mini prep method leaves too much RNA contamination. Alkaline lysis mini prep that includes additional steps to remove RNA e.g. RNase digestion followed by phenol extraction and isopropanol precipitation will give better results.
Alkaline lysis/PEG precipitation for large scale preps (see Maniatis Cloning Manual or similar), DNA must be free of PEG and salt!

LARGE DNA: In order to sequence large DNA templates such as BACs (bacterial artificial chromosomes), bacteriophages, cosmids and fosmids what is essential for successful sequencing is that the DNA must be free of any contaminants, including RNA and salts (see Contaminants) since a large amount of DNA must be used. We suggest using standard alkaline lysis, followed by RNase treatment, phenol:chloroform extraction and isopropanol precipitation, ethanol wash in order to isolate high-quality DNA.

DNA Sample concentration

Depending on the nature of the DNA different DNA concentrations are suggested:

*These reactions are more expensive (see Prices), and usually yield 100 to 300 bases of sequence.

Turnaround Time

Turnaround time is usually 48 hours (Weekdays only) from the time samples arrive at the laboratory.

Sequencing Primer Design

For the best results locate your primer at least 40 bases away from the region of interest. If you are using the same primer for PCR and for sequencing make sure that there are no dimers generated in PCR, as the dimer region of the sequence will not be readable.
Make sure that there is a single annealing site for your primer. Check the Troubleshooting section.
Primers should be 18 to 25 basepairs in length
Tm of primer should be at least 50 ºC at 2.5 mM MgCl2 and 0.2 mM primer
G-C content should be between 35% and 65%, 50% would be the best.
Avoid hairpin loops and long (more than 3) runs of any base.

DNA Sequencing reaction procedure

We use the ABI Big Dye terminator cycle sequencing chemistry to perform DNA sequencing. Conditions for the standard sequencing reaction are: annealing temperature 50 °C, extension 60 °C, 2.5mM MgCl2 and primer concentration is 0.2 mM. After the completion of the sequencing reaction, excess dye terminators are removed using Edge Bio Systems Performa DTR V3 96-Well Short Plates (Cat.# E-80808). The purified reaction is placed in the 3730 DNA automated sequencer.
On the Machine:
The capillary electrode is immersed in the DNA sample, current is applied, and the negatively charged DNA fragments enter the capillary by a process called electrokinetic injection.
Any residual reagents and contaminants will also enter the capillary, occasionally causing the sample to run in an abnormal way.
It is imperative that the DNA is very clean or the reaction might fail.
The capillary is then moved to the running buffer and the DNA fragments then migrate towards the detection window and the fluorescence of the di-deoxy-terminating base is measured, the software then determines the base.

New DNA basecalling software LongTrace is now available. This new basecaller significantly improves the read lengths of traces collected on ABI sequencers including ABI 3730 used in Mobix lab.