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Real-Time PCR and High Resolution Melting Curve Analysis (HRMA)
System
The Leiden Genome Technology Center has several systems available for Real-Time PCR and HRMA. Available equipment:
- Applied Biosystems Taqman 7900HT (Real-Time PCR and end-point SNP analysis), with a robotic arm to load plates. 96- and 384-well, and Taqman Low Density Array (TLDA) block available. More information is available on the Applied Biosystems website.
- Roche LightCycler 480 (Real-Time PCR, HRMA, and end-point SNP analysis), 96-well and 384-well block. See the Roche website for more information.
- Fluidigm Biomark: Real-Time PCR, expression analysis, SNP-genotyping, and digital arrays that are especially suited for detection of minimal residual disease and absolute quantification. The system includes a Nanoflex IFC controller 48*48. We also have access to a 96*96 loader via our collaboartion partner. At the moment we are involved in an early access project for the Access array loader. More information on the Biomark is available on the Fluidigm website.
- Idaho Technology LightScanner (HRMA only). In only 10 minutes a 96-well plate can be analyzed. More information on the Lighscanner can be found on the Idaho Technology website .
All additional information on the equipment, technology, assays, probes, and dyes can be found on the above mentioned web pages.
Real-Time PCR and HRMA at the LGTC
Projects
When you have a project for Real-Time PCR and/or HRMA and want to discuss it with us you can make an appointment with the LGTC. We can help you evaluate the possibilities, costs, planning, and all other issues. For large projects or series of samples of bad sample quality it may be wise to start with a pilot experiment.
First time users
When you plan to use the Real-Time PCR and HRMA machines for the first time please make an appointment via the LGTC for a training.
Equipment is free to use
The Taqman 7900HT, the Lightcycler 480, and the Lightscanner are free to use. We charge for the use of the Fluidigm, 48*48 loader and the access array. Please contact us for the prices for the use of the 96*96 loader. Provide your own plates and master mixes except for the Fluidigm. For the Fluidigm Biomark we ask you to order the arrays via us so that we try to get bulk discounts from the manufacturer.
User list
Please fill in the user list next to the machines, once is enough. Keep this list up to date. We use it to inform the users about updates, problems, maintenance, and all other issues which can be of importance in the projects.
Quotation (Fluidigm projects only)
Once it has been decided how to start with your Fluidigm project, you will need a quotation from the LGTC. This quotation can be requested via the LGTC order system for which you will need a valid account. A quotation for Fluidigm experiments contains the following products: Fluidigm order charge, Fluidigm arrays (digital, dynamic, or access arrays), and reagents needed for the loading of the samples and the arrays. It also must contain the charge for the use of the Fluidigm, the loaders, and/or the access array. Specific rules / prices apply to the 96*96 loader. When you have a Fluidigm project followed by next-generation sequencing it also has to be included in the same quotation. To accept the costs you need to deliver a printed and signed version of the quotation to the LGTC. Only then, the project will start.
Planning
Once you have been trained on the systems by a LGTC technician, the planning is up to you. Book your time in the Taqman 7900HT or Fluidigm Yahoo calendar. Please fill in your name, phone number, type of plate and run, and if you plan to use the robot. Do not reserve more time than needed.
The Lightcycler 480 has a paper agenda next to it and the same rules apply as for the Yahoo calendars.
The Lightscanner does not have an agenda since the HRMA only takes 10 minutes.
Data and data analysis
You are responsible for the data management of your experiments. We regularly delete data from the PCs due to lack of disk space, it is your responsibility to make your own back ups.
The LGTC has a data-analysis PC available for Fluidigm users. The latest version of analysis software is installed.
More information? Questions?
Contact the LGTC.
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