Skip to main content

Chemistry guides and trouble shooting

Primer availability
We offer the following primers for use in sequencing reactions.

Chemistry guides and trouble shooting

Primer availability
We offer the following primers for use in sequencing reactions.

 

Primer Sequence (5'-3') Primer length
M13-F CAC GAC GTT GTA AAA CGA C 19
M13-R GGA TAA CAA TTT CAC ACA GG 20
SP6 GAT TTA GGT GAC ACT ATA G 19
T7 TAA TAC GAC TCA CTA TAG GG 20


Sequence reaction protocol
Please note: 1/8 dilutiuon 10 μl reactions are the standard reaction performed 5.0  pmol primer is standard.

  • Prepare primers to 5.0uM (5.0pmoles/ul)
  • Calculate volume of template required from table below.
Template length DNA volume
100-200bp 1-3ng/µl
200-500bp 5-20ng/µl
1000-2000bp 10-40ng/µl
>2000bp 20-50ng/µl
ssDNA plasmid 25-50ng/µl
dsDNA plasmid 50-300ng/µl
Cosmid/BAC 500-1000ng/µl
Bacterial genomic DNA 2000-3000ng/µl


Alternatively, with 3µl pcr product loaded on gel, 1 μl of a sharp bright band is usually sufficient to produce good sequence.

  • Make a master mix of your sequencing reaction based on the following volumes:
Template length DNA volumne
2.5X Big dye terminator 0.5µl
5x Sequencing buffer 2.0µl
5µM primer 1.0µl
Template x µl (as above)
PCR grade water to 10µl
  • Aliquot mastermix to 0.2ml Eppendorf tube and add template
  • Run on ABI9600 (or equivalent thermal cycler) at:
Speed Temperature Time
1x 96 degrees 1 minute
25x 96 degrees 10 seconds
50 degrees 5 seconds
60 degrees 2 minutes*
Hold 4 degrees

* Extension time can be altered as for pcr protocol, i.e.  ≤ 1000 bp/min. Products are stable at 4ºC for several days or at -20ºC for several weeks.

Reference:
DNA Sequencing by Capillary Electrophoresis Applied Biosystems Chemistry Guide Second Edition
Additional protocols for purifying and quantifying DNA products prior to sequencing or genotyping are also available in this document.

Sequence reaction cleanup protocol
We use the Agencourt CleanSEQ magnetic bead-based sequencing purification system to remove unincorporated dyes, nucleotides, salts, and other contaminants after the sequencing reaction. If you have used or wish to use a different protocol please inform us when you submit samples.

For more information see AGENCOURT® CLEANSEQ® Dye-Terminator Removal.

Genotyping (Microsatellite/Fragment Analysis)
Fragment analysis generates a size estimate for DNA fragments relative to a size standard of DNA fragments with known lengths. The size standard is combined with the sample of interest and co-injected on the capillary electrophoresis system.
An overview of the genotyping workflow is available on the Applied Biosystems website.

Details of the chemistry are available online at Genescan®.

Trouble shooting
There are many reasons for a failed or poor quality result. We recommend you review the electropherogram, annotation and raw data for each sequence, using programmes such as Sequence Scanner, Sequence Analysis or Chromas to import the .ab1 files.

Troubleshooting information for fragment analysis data is available in GeneMapper® Software Version 4.1 Reference and Troubleshooting Guide.

Additionally, we are always willing to address your queries. Email us at sequence@lincoln.ac.nz