Mouse Genotyping

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Sample Submission Guidelines

New Project Submission Form

Mouse Genotyping Client Questions and Answers 


Mouse Genotyping at Garvan Molecular Genetics

 

 

Mouse Genotyping WorkflowIn collaboration with Australian BioResources (ABR) in Moss Vale, Garvan Molecular Genetics offers a mouse genotyping service. Rodents are bred and clipped at ABR, after which DNA is prepared with an automated liquid handling system.

The isolated DNA is then sent via shuttle to Garvan Molecular Genetics, where high throughput fully automated mouse genotyping is performed. External clients can also send their DNA or tissue to Garvan Molecular Genetics directly for this service.

We use a combination of realtime PCR and meltcurve analysis similar to Jackson Laboratories in the US to deduct the genotypes of the DNA samples. Results are sent to clients via email.

This service is NATA certified (ISO 17025).

 

Sample DNA quality

The analysis method of our Mouse Genotyping protocol is sensitive to the quality of the DNA that we are given. This is due to the fact that we run a realtime PCR with a program suitable for most primer annealing temperatures. We have observed that purified DNA gives the best results. The following graphs show the effect of different DNA preps:

DNA Prep Graphs

We recommend to clean the proteinase K digestion via columns or magnetic beads. Any Qiagen or similar cleanup kit will do a great job. We will accept DNA that has not been treated in the recommended way but we must make you aware that we observe a dropout rate of ~20% of samples. Also, there is an increased risk of genotyping errors due to higher salt concentrations or inhibitors. The meltcurve analysis is highly sensitive to the salt concentration and variation of the salt concentration shifts the peak of the genotype relative to the controls.

With the DNA preparation service at ABR in Moss Vale the DNA quality equals column cleaned DNA. After the genotyping process the DNA can also be sent if you needed to perform further downstream applications.

 

Establishing New Mouse genotyping Projects

We will establish your current mouse genotyping PCR protocol on our system for $120. We can waive this fee depending on expected sample numbers that will be sent in future. Please fill in the New Project Submission Form. Please tell us to whom to report to, the gene name,  the primer sequences, the primer combinations & product sizes, and expected genotypes (eg wildtype, heterozygous, homozygous). We will also need some controls (HET, WT, HOM) to get us started. These controls can be DNA samples with known genotypes (confirmed by previous genotyping) or tissue from animals with known genotypes (eg founder animals).

We will order the primers and will establish the project once primers and controls have arrived. In case you have no primer sequences and know only the gene name or gene sequence we can also design primers for your genotyping. We will be in contact with you along the way and inform you about the progress. 

In case time matters and you need us to establish your project very quickly, you can provide us with primer aliquots and we will start genotyping immediately for you.

Whilst it is the responsibility of each client to submit the correct primers (or sequence file to design primers) when establishing a new project we want to assist the process and make available our database of all existing primers and PCRs that are currently established at GMG. The document GMG Mouse Genotyping Existing PCRs and Primers contains three Excel sheets. In the Excel sheet called "MGS Primers" you can check whether we already work with a primer that you intend to use. The primer number in column A (4 digits) is used in the Excel sheet called "Genotyping Information" and can identify an already established PCR. Finally, in the Excel sheet called "Line Information" we associate the PCRs with mouse line names at ABR. If you identify a mouse line name in this sheet and click on the blue genetags they will be linked to the PCRs (and primers) used for these lines. Please ask us if you need more information.

 

Sample Submission

For routine genotyping we prefer samples to be in a 96 well format as these plates fit into our robotic liquid handlers. These skirted 96 well PCR plates incl. seals can be purchased through us or via Biorad directly (BioRad plates HSP-9901 & Microseals MSB-1001). Please read our  Mouse Genotyping Service Sample Submission Guidelines and Mouse Genotyping Client Questions and Answers before submission.

 

Additional Positive Controls

Some researchers prefer to add positive controls to each plate they are sending to us and for each test they want us to perform. We greatly appreciate this as it adds additional safety to the tests. If you can, please add positive and negative controls to your sample submission plate(s).

 

Personal Sample Delivery

How to submit your samples is decribed in detail in our Mouse Genotyping Service Sample Submission Guidelines. The volume of the sample DNA should be at least 30ul in each well of the 96well plate with a concentration of at 5ng/ul for standard genotyping. For copy number analysis we need at least 100ul with 50ng/ul concentration of the submitted DNA. We will not use all of the submitted DNA, we will most likely only use 1ul of this DNA but we need this volume for the robotic liquid handlers to safely operate. After genotyping results have been sent you can pick up the remaining DNA from the sample reception fridge if you needed the samples for further testing. The 96-well sample plate for genotyping can be placed into the first shelf of the “Mouse genotyping reception fridge” which is located on level8 in room 8.01 and has the asset number #5155. Please place your samples here and send an accompanying email with the Mouse Genotyping Sample Submission Form to 

 

Sample submission via ABR in Mossvale

Contact ABR Scientific Services Manager Kevin Taylor (02 9295 8596 / k.taylor@garvan.org.auto arrange for your samples to be processed in Moss Vale and sent directly via shuttle, with no further action required on your side.

 

Sample submission via Australian Post

You can also send your samples dried down after Ethanol precipitation in a 96 well plate with seal via Australian Post to: 

Garvan Molecular Genetics
Level 8 Garvan Institute
384 Victoria Street
Darlinghurst, NSW, 2010

Also, please send a Mouse Genotyping Sample Submission Form to our email address at  with the billing information and an email address to which the results should be forwarded.

 

How we genotype your sampleMouse Genotyping how we do your genotypoe

We perform a touchdown realtime PCR in a 384well plate scale. Samples are processed via liquid handlers and analysed on our LightCycler 480 with a Sito9-based Meltcurve analysis. The control meltcurve peaks are compared to the sample peaks. The test format is also used by the Jackson Laboratories in the USA. We run positive and negative controls for each test and only release results when all controls have been validated.

 

 

Pricing

For prices please see our Molecular Genetics Shop website.

 

Getting your results

The results of the genotyping will be sent to you via e-mail. We usually complete the genotyping within 48-72 hours after receipt of sample. In case we have dropouts we will repeat them once. If the genotype can still not be determined we will inform you about the problem. In exceptional cases the genotyping might be delayed by a day if instruments are out of order or staff is sick.

You can also label your samples as urgent and we will make a special effort to process them ASAP. Samples can be analysed with highest priority within 8 hours after receipt of sample.

 

Additional services

Copy number analysis (Het/Hom differentiation)

When exactly the same amount of DNA is added into a PCR reaction and the amplification process monitored via realtime PCR a homozygous sample will have a crossing point of one cycle earlier than a heterozygous sample. Therefore we can distinguish heterozygous samples from homozygous samples for any given gene by this protocol. For the method to work we quantify the DNA exactly, dilute to the same concentration and set up 5 PCRs (repetitions) of each sample and 2 house keeper genes, amplify in realtime mode and form the average Ct value for each sample, subtract the house keeper and compare to controls. This test only works with very clean DNA and a het and hom control.

Het/Hom differentiation 

High Resolution Meltcurve (HRM) Analysis

SNP analysis

We have developed a reliable and very price competitive method to type SNPs in a high-throughput format via High Resolution Meltcurve (HRM) Analysis.

Please mail the samples to Garvan Molecular Genetics via Australia Post (see address above)

 

Mouse SNP panel for genetic monitoring of inbred strains

We also offer a 28 SNP panel for quality controlling your inbred mouse lines routinely. The panel assesses genetic contamination in colonies containing a large number of genetically diverse mouse strains. This test is used by the Jackson Laboratory for their mouse lines and investigates 28 relevant loci which cover over 300 inbred, wild-derived, congenic, consomic and recombinant inbred strains.