The Center bases its work on the following instruments:

• StepOnePlus™ Real-Time PCR Systems (https://www.thermofisher.com/order/catalog/product/4376600)
• QuantStudio 3K
• QuantStudio 12K Flex Real-Time PCR System (https://www.thermofisher.com/il/en/home/life-science/pcr/real-time-pcr/real-time-pcr-instruments/quantstudio-12k-flex-real-time-pcr-system.html)
• Fluidigm BioMark™ HD System (https://www.fluidigm.com/products/biomark-hd-system)

The available applications:

• Real-Time PCR using the SYBR Green I chemistry
• Real-Time PCR using the TaqMAN Probe
• SNP (Single Nucleotide Polymorphism) detection using the TaqMAN Probe
• High-throughput (96x96) RT PCR on Fluidigm BioMark.

Consumables and Disposables

• ABI PrismTM 96-well Optical Reaction Plates sealed with Optical Adhesive Covers
• ABI PrismTM 384-well Optical Reaction Plates sealed with Optical Adhesive Covers
• Micro Fluidic Cards

Real-Time PCR using the SYBR Green or TaqMAN:

There are three options:

1. Running a 96-wells PCR plate, the assay is done by the customer.
2. RNA samples are submitted. RT-PCR amplifications and Real-Time PCR are performed at the Center, with primers provided by the customer.
3. cDNA samples are submitted. Real-Time PCR is performed at the Center, with primers provided by the customer.

Requirements for Primers (SYBR-GREEN chemistry)

• From 30 to 80% G/C content.
• Melting temperature (Tm) from 58 to 60°C.
• The five nucleotides at the 3´ end of primer should have no more than two G or C bases.
• Recommended primer concentration 50nM.  
• Length of amplicons should be in 50-150 basepairs range.

Requirements for Primers and Probe (TaqMAN chemistry)

• Primers and probe with 30 to 80% G/C content
• Melting temperature (Tm) for the primers 58 to 60°C, Tm for the probe 8 to 10°C higher.
• Do not select probes with G on the 5´ end.
• The five nucleotides at the 3´ end of primer should have no more than two G or C bases.
• Recommended primer concentration 900nM and probe concentration 250nM.
• Amplicons are in the 50- to 150-basepairs range.   

SNP (Single Nucleotide Polymorphism) detection using the TaqMAN Probe

SNP Assays provided by the customer.

Genomic DNA samples are submitted

The DNA concentration should be 10-20 ng/µl.

The volume of the samples depends on the number of requested SNPs but not less than 20 µl.

DNA samples should arrive in a 96-wells plate even if the plate is not full.

The order of the samples in the plate should always be as follows:

          Sample 1 is in position A1

          Sample 2 is in position B1

          .....................................

          Sample 96 is in position H12

High-throughput (96x96) RT PCR on Fluidigm BioMark.

Real-Time PCR is performed at the Center.

IFC-array provided by the customer.

For Gene Expression analysis the customer need to provide plate with cDNA samples and plate with diluted primers. The final concentration of Forward+Reverse primers should be 50µM.