Genetic Data Centre - University of British Columbia
   
   

  

   

Methods

Researchers at the Genetic Data Center select the best method for achieving the results required. Some of the methods we use on a regular basis are:

Isolation/Extraction

Many isolation techniques are used in the G.D.C. and each organism will be customized as to the best method for DNA and Isozyme isolation. Some examples of organisms encountered in the G.D.C.are: conifers, annual and perennial plants, birds, fish, insects, mammals.

DNA Isolation

Grinding plant tissues with liquid nitrogen

Isozyme Extraction

Grinding samples using tissue homogenizer

Markers

PCR based markers

Sequencing and Genotyping (SSRs, AFLP etc.)

MJ thermal cycler

Sequencing

The DNA Sequencers employed by the G.D.C. are the LI-COR® 4200 series automated sequencers. The capacity is 16 samples per channel for sequencing.

Sequencing Reaction

For the sequencing reaction, the DNA polymerase incorporates an infrared dye (IRD)-labeled primer into a set of chain-terminated fragments or the tailed sequencing method; the latter is the most commonly used method in the G.D.C. (see Steffans et al, Biotechniques 15:580-581, 1993). The IRD-labeled fragments separate according to size on a polyacrylamide gel. IRD labeled primers are sensitive to visible light. (Avoid extended exposure of IRD labeled primers to light by storing in the dark and wrapping primer stock tubes in foil for use on the benchtop.) Upon completion of the run, the sequence data can be presented as text (ASCII) or in curve format (standard chromatogram format, SCF) or as bands (tag image format, TIF).

Two different SequiTherm EXCELII Long-Read Sequencing Kits are available for use on LI-COR® Automated DNA Sequencers.

  1. "SequiTherm EXCEL™ II DNA Sequencing Kit-LC (for 66-cm gels)" This kit has termination mixes which permit read lengths up to 1000 to 1400+ bases.
  2. "SequiTherm EXCEL™ II DNA Sequencing Kit-LC (for 25-41-cm gels)" This kit provides darker signal intensities in the resolution range of 1-1000 bases. Users are those who routinely sequence templates of smaller size, such as PCR products or clones for verification.
Sequencing GDC
Sequencer Li-Cor® 4000 and 4200 automated sequencers
Channel IRD700 and IRD800
PCR Machine MJ thermal cyclers
Chemistry SequiTherm EXCEL™ II DNA Sequencing
Sequencing Enzyme SequiTherm EXCEL™ II DNA Polymerase
Primers M13 tailed/end-labeled primers
Accuracy greater or equal to 99% over 1000 bp
Template Material ds DNA from plasmid (250-500 ng per reaction)
  PCR product (125-250 ng per reaction)
Template Preparation Wizard® PCR Preps DNA Purification (for PCR products if necessary)
Primer Concentration 1-2 pmol
Range 75 to 1500 bp
Removal of excess terminations Not required when using the tailed method
LiCor machine recovery Size product >900 bp (66cm)
  Size product <900 bp (66cm)
  Size product <650 bp (41cm)
  Size product <450 bp (25cm)
LiCor data collection and scoring e-Seq™ and AlignIR Alignment and Assembly Software™

All trademarks are property of their respective owners.

Product information, protocols and references can be obtained from the following web sites:

Sequencing from plasmids,

Sequencing directly from PCR products and Others:

Sequencing from dsDNA from plasmids

The quality and quantity of DNA templates is crucial in sequencing reactions. In terms of template added, please note that more is not better; excessive amounts of DNA in the sample can result in poor resolution, streaking, and lowered signal intensity or no readable signal. However, too little template will also result in poor resolution or premature termination of the sequence.

The SequiTherm EXCEL™ II kits are designed for plasmids, PCR products, and M13-based templates. Cycle Sequencing protocols are available at http://www.interscience.com/ and http://www.licor.com/. Larger templates like BACs, cosmids, and lambda phage can be sequenced with modifications in template concentration and number of cycles.

Sequencing PCR product directly

PCR products can easily be sequenced directly using the tailed primer method outlined above. The PCR product should be tested on an agarose gel and appear as a single band without any artifacts. Single band PCR products are the best templates for DNA sequencing. If most of the PCR primers are incorporated, the amplification product can be sequenced directly without purification. The PCR product can be purified by one of the following recommended methods:

  • Wizard® PCR Preps DNA Purification
  • QIAamp DNA Mini-kit

All trademarks are property of their respective owners.

Product information, protocols and references can be obtained from the following web sites:

Please contact us for other options such as:

Asymmetric DNA

Simultaneous bi-directional sequencing

 


 

 
Department of Forest Sciences - U.B.C. Vancouver, B.C. - V6T 1Z4 - Tel (604) 822-3908 or (604) 822-1543 - Fax (604) 822-9102