Mouse Genotyping at Garvan Molecular Genetics
Garvan Molecular Genetics offers a rodent genotyping service for mice and rats. DNA is extracted from submitted tissue samples and extracted via our high throughput robotic platforms. PCR is set up with fully automated liquid handling systems that are controlled by our in-house developed programming software.
We use a combination of realtime PCR and High Resolution Meltcurve analysis to deduct the genotypes, similar to Jackson Laboratories in the US. Results are sent to clients via email. DNA samples are stored for 6 months and can be sent to clients for further experiments upon request.
We are committed to providing an excellent service quality. Therefore, we aim at continuously improving the service to achieve highly reliable results, short turnaround time and minimal costs. Client satisfaction is monitored by annual surveys and constant feedback.
This service runs in a quality controlled environment according to ISO standard 17025 as certified by NATA.
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:
If you send us DNA samples rather than tissue we recommend cleaning 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.
Our DNA extraction from tissue uses spin columns from Qiagen to prepare the DNA. This guarantees the highest possible quality DNA for genotyping and downstream applications. Please send us between 3 to 5mm of tail tissue or alternatively at least 3mm2 of earclip tissue.
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 (see top of page). 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 your mice are bred there). 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.
For routine genotyping we prefer samples to be in a 96 well format as these plates fit into our robotic liquid handlers. we can send you special plates for tissue or DNA samples, please send us an email. All information in regards to sample submission is explained in our Mouse Genotyping Service Sample Submission Guidelines and Mouse Genotyping Service Questions & Answers (see top of page).
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 described in detail in our Mouse Genotyping Service Sample Submission Guidelines (see top of page). If you submit DNA samples the volume of the DNA sample should be at least 30ul in each well of a 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. If you submit tissue samples please make sure they are between 3 - 5mm of tail tissue or alternatively at least 3mm2 of earclip tissue.
Sample submission via Australian Post
You can also send your samples via Australian Post or courier to:
Garvan Molecular Genetics
Level 8 Garvan Institute
384 Victoria Street
Darlinghurst, NSW, 2010
Also, please send a Mouse Genotyping Sample Submission Form (see top of page) to our email address at email address with the billing information and an email address to which the results should be forwarded.
How we genotype your sample
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.
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 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 for an additional service fee.
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.
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.