Core Facilities

3100 and 3130xl Genetic Analyzer Information

Baker IDI has a 3100 and a 3130xl Genetic Analyzer, an automated, high through put, capillary electrophoresis system used for analyzing fluorescently labeled DNA fragments.

The presence of RNA, protein, residual detergents and residual salt can interfere with electrophoresis and electrokinetic injection, which may then shorten the life of the capillaries and therefore affect results.

Samples must be checked on a gel and Spec (260nm) for accurate concentrations and/or contamination. Samples must be re-suspended in water only! Column purified DNA samples get better results. (Suggested columns are Qiagen, Bresatec and Wizard).

The chemistry used is Big Dye Terminator Chemistry v3.1. This chemistry is recommended for the majority of sequencing applications. If you have a difficult, or other template please check before you prepare your samples if this chemistry is suitable. You may prepare your own samples for sequencing and supply a dried sample, but you must use Big Dye Terminator Chemistry v 3.1.

Recommended Concentrations of DNA and primers

Primers at 3.2pmol/μl

You Must:

• Supply enough DNA for each reaction (don't just add 1ml to the tube)
• Supply a primer that is suitable for the template and at the correct concentration
• Dilute your samples within the ranges specified ie: Double Stranded 150-300ng
• Write down the concentration of your DNA (DO NOT GUESS)
• Fill out the request form correctly (ie: one line for each reaction, including different primers). This includes your name, Lab name, order number, date, concentration of DNA and legible hand writing. Please keep template/file name to a minimum of 15 characters.
• Label tubes correctly and include your name and date.

Primer design and purification can affect the quality of sequencing data.

For best results:

• 18-22 bases long
• Avoid runs of identical nucleotides (eg 4 x G's)
• G-C content 30-80% (pref. 50-55%)
• Tm > 45oC
• Preferably purified by HPLC

Please note that samples must be in by 4.30pm the day before the run. Runs for Sequencing and Electrophoresis samples are conducted on Mondays and Thursdays, and each weekday for Electrophoresis only samples. Turnaround time for results is 24-48 hours.

External DNA sequencing request form

External Electrophoresis Only Request Form 

DNA Sequencing by Capillary Electrophoresis Chemistry Guide

Any queries can be emailed to the sequencing lab: dnasequence@bakeridi.edu.au or 8532 1230