Genotyping

Microsatellites (SSR)

The DNA Sequencers employed by Genetic Data Centre are LI-COR® 4200 series automated sequencers. The capacity is 128 samples per run (64 samples per channel) for SSR gels.

Preparing Genomic DNA

Most DNA preps will work, but the DNA should have a 260/280 ratio of 1.7.

Preparing Primers

Primers for genotyping can be infrared dye (IRD) labelled from LiCor. M13-tailed primer is a lower cost alternative to custom labeled primers. One of the unlabeled STR primers is synthesized with an M13 forward or reverse primer sequence on the 5'-end and is used in combination with another primer without the tail. An IRD-labeled M13 Primer is included in the PCR reaction (see Oetting et al, Genomics 30:450-458, 1995). The M13 primer is added to the PCR product during the first few cycles of amplification. The labeled M13 primer is incorporated in subsequent cycles, thus labeling the PCR product. These labeled PCR products are sensitive to visible light and it is necessary to avoid extended exposure of IRD labeled primers and PCR products to light by keeping them stored in the dark and wrapping PCR racks/plates in foil for use on the benchtop.

Size Standards

The STR Marker is composed of IRD-labeled DNA fragments with equal banding intensities in 90% formamide solution with bromophenol blue. Then fragments cover the size ranges of 50 to 350 bp and 50 to 700 bp (both marker ladder sets are available).

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Product information, protocols and references can be obtained from http://www.licor.com/.

Amplified Fragment Length Polymorphism (AFLP)

The capacity is 128 samples per run (64 samples per channel) for AFLP gels.

Preparing Genomic DNA

High quality of geno DNA is required; the DNA should have a 260/280 ratio of 1.7 - 2.5. Large quantity of genomic DNA is also required (2 - 4 ug per sample).

Preparing Primers

AFLP® Analysis System 1 kit can be purchased from Life Technologies. An AFLP® primer testing kit is available at the G.D.C. Arrangements can be made with the director to test existing AFLP primers in the G.D.C. You can also make your own AFLP custom primers. The labeled AFLP products are sensitive to visible light and thus, it is necessary to avoid extended exposure of AFLP products to light by keeping reactions stored in the dark and wrapping PCR racks/plates in foil for use on the bench. No purification prior to gel analysis is required.

All trademarks are property of their respective owners.

Product information, protocols and references can be obtained from

http://www.lifetech.com/ and http://www.licor.com/.

Randomly Amplified Polymorphic DNA (R.A.P.D.)

Different electrophoresis units are available in the GDC. The capacity for gel electrophoresis is 60 samples per gel.

Preparing Genomic DNA

High quality of genomic DNA is required; the DNA should have a 260/280 ratio of 1.7 - 2.5.

Preparing Primers

RAPD primer kits are available from the N.A.P.S. Unit at the University of British Columbia.

Isozymes

The capacity for gel electrophoresis is 30 samples per gel, 8-12 gels per day.

Steps:

1. Tissue homogenization
2. Preparation of starch gels
3. Gel loading
4. Electrophoresis
5. Gel slicing
6. Histochemical staining
7. Scoring by eye or photodocumentation of results

Chemicals can be purchased directly from our suggested vendor, Sigma-Aldrich, or from the GDC.

Link:

http://www.sigma-aldrich.com/

Other markers (Contact us for more information)

• minisatellites etc.

Other techniques: (Contact us for more information)

• Cloning
• RT-PCR
• RFLP

Data Analysis (Contact us for more information)