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ABI Genotyping Service Technical Information
- Sample Requirements
- Fluorescent Labelling of Primers
- Primer Design Recommendations
- Genotyping Technical Information
- Microsatellite Fragment Analysis technical Information
The Massey Genome Service requires customers who are using the ‘Genotyping Capillary Separation Service’, to please provide your pooled reactions in a volume of 1ul, in either 0.2ml individual PCR tubes or 0.2ml strip tubes. For the ‘Genotyping Plate Service’, please provide your pooled reactions in a volume of 1ul, in a 96 well plate, sealed with strip tubes, plastic seal or foil seal. The strip tubes provide the best seals.
It is recommended that when you first set up your experiment to carry out a concentration titration series on your pooled genotyping products to determine optimal loading. Please contact Richard Fong for further information regarding optimising loading of products.
For genotyping with the Massey Genome Service customers use 5’-end labelled primers. Customers can use the following 4 ABI Prism® dyes to label their primers with:
- 6-FAM™ - Blue
- VIC® - Green
- NED™ - Yellow
- PET™ - Red
The Massey Genome Service uses GeneScan™ - 500 LIZ™ Size Standard, as the internal standard for fragment sizing. This size standard will accurately size fragments between 50-500bp, and provides 16 single-stranded labelled fragments of 35, 50, 75, 100, 139, 150, 160, 200, 250, 300, 340, 350, 400, 450, 490, and 500 bases. Each of the DNA fragments is labelled with a proprietary fluorophore, which results in a single peak when run under denaturing or native conditions.
NOTE: Customers do not have to provide the size standard. The Massey Genome Service will add the size standard to your samples before running. When designing your primers, please design them to produce fragments >75bp and <490bp. The Massey Genome Service uses ABI Prism® GeneMapper™ Software which looks for two size markers smaller and larger than the fragment being sized, in order to get an accurate size calling. The 35bp size standard fragment can sometimes be inaccurate because the unincorporated primer peaks obscure its detection by the software. Also the 250bp fragment peak in the GeneScan™ - 500 LIZ™ Size Standard, is not used in fragment sizing under denaturing conditions, due to the abnormal migration of double strands that did not completely separate under these conditions. The 250bp fragment peak frequently runs at 246bp, so it is not included in size calling. So there is a gap of 100bp between the 200bp and 300bp size standard peaks when sizing fragments in this size range.
The following recommendations are provided to help optimize primer selection:
- Avoid >2 GC’s in last 5 nucleotides at 3’ end of primer.
- Primers should be at least 18 bases long to ensure good hybridization.
- Avoid runs of an identical nucleotide, especially runs of four or more Gs.
- Keep the G-C content in the range 30-80%, preferably 50-55%.
For cycle sequencing, primers with TM>45°C produce better results than primers with lower TM.
- For primers with G-C content less than 50%, it may be necessary to extend the primer sequence beyond 18 bases to keep the TM>45°C.
- Use of primers longer than 18 bases also minimizes the chances of having a secondary hybridization site on the target DNA.
- Avoid primers that hybridize to form dimers.
- Avoid palindromes because they can form secondary structures.
- The primer should be as pure as possible, preferably purified by HPLC.
- To ensure specificity of primer (BLAST search) to the target.
- Dissolve primer stocks in 10mM TE buffer, pH8.0. But dilute working primer solutions in water because salt can affect primer extension.
Please download the attached ‘Genotyping Technical Bulletin’ below, which contains information on the following:
- Experimental design considerations.
- Multiplexing to increase throughput
- 3’A Nucleotide addition
- Stutter products
- Primer design recommendations
Please download the attached ‘Microsatellite Fragment Analysis Technical Bulletin’ below, which contains information on the following:
- PCR Amplification.
- Control DNA.
- Preparation of PCR Products.
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Last updated on Friday 01 March 2019