Taqman Probe Chemistry
Taqman probes used to be 25 to 27 nucleotides long and contained TAMRA as the Quencher. In old publications you might still find these fossils. To improve the detection of SNPs and the overall performance of the probes the overall length of the probe needed to be reduced without compromising on the binding efficiency of the probe to the DNA. The length of the probe determines its annealing temperature, which needs to be significantly higher than that of the primers. ABI invented the MGB domain (Minor Groove Binding domain) which is attached to the probe via a flexible linker and increases the binding to the DNA so efficiently that modern probes are in average 13 nucleotides long.
The assay will be delivered premixed (primers and probes) and no optimization regarding run conditions is needed. there is a guarantee for the assay to work (if problems arise technical support can be contacted). The assays can be ordered in various sizes and in different locations of the target gene (exon or intron spanning etc) please see below for more information. Thermofisher provides an online assay design on their website. Once on the webpage you can enter the organism you are working with and enter a gene ID, symbol or name or even a sequence and click on search to find appropriate Taqman assays. The online assay design webpage looks like the image below. In the document "How to find an inventoried Taqman probe" the steps of finding and identifying the Taqman assay are explained in detail.
Inventoried, Non-Inventoried Assays and Custom-MGB Probes
Inventoried Taqman Assays (also microRNA)
Depending whether an assay has been tested and is in stock or needs to be manufactured or needs to be designed and manufactured Taqman assays are classified as inventoried, non-inventoried or custom made. Inventoried Taqman assays have been tested and optimized and are stored on shelves in the warehouse. They are ready for shipping from the US and will be the quickest to arrive after ordering. Usually they take 1 to 2 weeks to arrive in the facility after they have been ordered. If you have ordered the Taqman assay via our Reagent Ordering Service we will notify you via email once the assay is ready for pickup in the facility.
Non-Inventoried (made-to-order) Taqman Assays (also microRNA)
If you cannot find an inventoried Taqman assay for your gene of interest there is still the possibility to use a non-inventoried (Made to Order) Taqman probe. Non-inventoried means that this assay theoretically works, the algorithms of the Thermofisher assay design center have calculated it should work. There is no guarantee though that is works, it has to be synthesized specifically for your order (that means it doesn’t come off the shelf). Therefore it will take longer (~3 to 4 weeks) and will cost more, though you will also get more assay volume.
Custom MGB Probes
Custom MGB probes are the last resort if your gene of interest is not yet catalogued (ie: not a commonly studied gene). These probes need to be manually designed and are expensive. Therefore please ask for help when designing these probes. There are guidelines within the ABI website for a step by step guide to design these probes. Please contact us and we will assist you in the design and ordering process.
Please find prices for Taqman assays on our Molecular Genetics Shop webpage.
Once you have identified the Taqman ID, please copy the assay number and name and paste it into our Reagent Ordering Form and send to our email address, see picture below. We order Thermofisher reagents once a week (on Wednesday) or if your order is urgent please tick the "urgent" box in the Reagent Order Form and we will order your assay on the day your order form is received. As we order in bulk Thermofisher has applied a significant discount to our account and you also save on individual shipping costs.
Once the assay(s) have been received in the facility you will be notified via email when the assays are ready for collection.
All assays will come at the following concentration:
- Delivered as 20X premix of primer and probe
- primer concentrations @ 900nM
- probe @ 250nM
- use 0.5ul of premix for a 10ul PCR reaction
Taqman Assay Nomenclature
The Taqman assays have Cataloge numbers that define their scale (for example 4331182 is a inventoried assay). The actual assay ID has two letters in the front to identify the organism followed by a 8 digit number followed by an underscore and an extension for example "Hs01548421_g1". The extension identifies the assays design, here is a list of what the extensions mean:
- "_m" indicates an assay whose probe spans an exon junction and will not detect genomic DNA.
- "_s" indicates an assay whose primers and probes are designed within a single exon. Such assays will, by definition, detect genomic DNA.
- "_g" indicates an assay that may detect genomic DNA. The assay primers and probe may also be within a single exon.
- "_mH," "_sH", or "_gH" indicates that the assay was designed to a transcript belonging to a gene family with high sequence homology. The assays have been designed to give between 1,000– 30,000-fold greater discrimination from the closest homologous transcript.
- "_u" indicates an assay whose amplicon spans an exon junction and the probe sits completely in one of the spanned exons.
Setting Up Your Realtime Experiment
We keep stocks of Taqman GE Master Mixes for you at the facility. TaqMan Gene Expression Master Mix, 5ml (PN: 4369016) for use with TaqMan probes (inventoried, non-inventoried and Custom MGB probes) and TaqMan Power SYBR Green PCR Master Mix, 5ml (PN: 4367659) for use in SYBR Green assays. We also have the necessary 384well microtiter-plates (MicroAmp Optical 384-Well Reaction Plate with Barcode) and Optical Seals in stock (Microseal ‘B’ Film PCR Seales), Cat#: MSB1001. For prices see our shop webpage.
There are two 7900HT instruments at the Molecular Genetics facility which can be booked on the booking system "GMG_7900_2831" or "GMG_7900_3091". The instruments are absolutely identical.
The software to setup and analyse the data is called SDS2.4 and can be downloaded from our server. The software only installs on Windows operating system.
Alternatively you can use the Lightcycler 480 or the Biorad C1000 instruments for running your 384 well plate. please ask us for help as the default run parameter need to be changed. For PCR setup in 384 well plates (the facility only holds 384 well PCR instruments) we strongly recommend to use the epMotion 5070 liquid handler robots. How to use the 7900HTs and the epMotion robots is explained in individual training sessions on our training ipad which can be booked via the booking system.