Real time PCR equipment

All Real-Time PCR technology projects can be done with Good Laboratory Practice (GLP) standard.

The Center bases its work on the following instruments:

The available applications:

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.

Microfluidic card (Low Density Microarray)

Real-Time PCR is performed at the Center, with card provided by the customer.

cDNA samples are submitted.

Concentration of cDNA between 25-50 ng/µl.

Amount of cDNA 100 ng/port (48 reaction chambers)

Always include Sample submission form for Cards.    

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.