FRET Probes

FRET probe



A LightCycler FRET probe system is a pair of single-stranded fluorescent-labeled oligonucleotides. They are sequence specific and highly sensitive.


FRET probes for Real-Time PCR can be ordered in our webshop or by email. Download our excel orderform for ordering by email or use it to upload multiple probes in our webshop.

Order your primes and probes delivered premixed according to your specifications. All fluorophore -quencher combinations are possible, ideal for Real-Time PCR applications.

With our oligo prepaid service we make oligo ordering easy. Credit can be easily transferred to your account. Oligos can then be ordered immediately, eliminating possible delays due to purchase authorization. Please contact Biolegio for more information.

You can fit your Real-Time PCR probe with numerous fluorophores and quenchers from an extensive list of modifications. 

>view fluorophores            >view quenchers
 
 

Quencher and dye compatibility

Please follow the link below for a compatibility figure. For multiplexing applications use fluorophores with the least amount of spectral overlap.

>Fluorophores vs Quenchers compatibility
 
  

How can I fit modifications to my custom Real-Time PCR probe?

Select the modifications of your choice in our webshop or in our excel ordering sheet. In the webshop you can have instant pricing insight. Select 'TDP probe' in the orderform as the chemistry used for ordering Twice Dyed Probes probes.
  

How can I add mixed bases to my oligo?

Use the standard code letters for mixed bases / wobbles

A lightCycler FRET probe system is a pair of single-stranded fluorescent-labeled oligonucleotides. Probe 1 (the donor probe) is labeled at its 3'-end with a donor fluorophore (generally fluorescein) and probe 2 (the acceptor probe) is labeled at its 5'-end with one of four available fluorophores (red 610, 640, 670 or 705). The free 3' hydroxyl group of probe 2 must be blocked with a phosphate group (P) to prevent Taq DNA polymerase extension. To avoid steric problems between the donor and the acceptor fluorophores on both probes, there should be a spacer of 1 to 5 nt (4 to 25Å distance) to separate the two probes from each other. before any real-time quantitative PCR reaction takes place, fluorescence background may be observed inside the tube.

During the annealing step of real-time quantitative PCR, the PCR primers and the LightCycler probes hybridize to their specific target regions causing the donor dye to come into close proximity to the acceptor dye. When the donor dye is excited by light from the lightCycler instrument, energy is transferred by Fluorescence Resonance Energy Transfer (FRET) from the donor to the acceptor dye. The energy transfer causes the acceptor dye to emit light at a longer wavelength than the light emitted from the LightCycler instruments optical unit. The increase in measured fluorescent signal is directly proportional to the amount of accumulating target DNA.

Biolegio provides alternative dyes for use on the LightCyclers.

All Real-Time PCR probes are delivered HPLC purified, this is included in the price. 

>more about purification

Real-Time PCR probe scale and length

Biolegio three different synthesis scales for Real-Time PCR probes oligos: 40 nmol, 200 nmol and 1000 nmol. Prices include synthesis of maximum 40 nucleotides.

Yield Real-Time PCR probes

40 nmol scale:    approx. 8 nmol min. yield
200 nmol scale:   approx. 20 nmol min. yield
1000 nmol scale: approx. 60 nmol min. yield

Method

Biolegio has three synthesis protocols: 'Economy' , 'High Purity' and 'B-pure'. The used protocol depends on the length of the oligo. This means that the protocols have been optimized for certain oligo lengths. 

  • Economy protocol is used for oligos between 2 and 49 bases.
  • High purity protocol is used for oligos between 49 and 79 bases. 
  • All DNA oligos longer than 80 bases are synthesized on our B-pure synthesis protocol.

Each protocol delivers the highest possible coupling efficiency for the specified range. 

Your Real-Time PCR probe can be shipped dry or desolved or mixed. Standard probes are delivered desolved in a tube in a concentration of 100 pmol/ul.

Available buffers
  • H20 buffer
  • LoTE buffer
  • TE buffer
     
Your primers and probes delivered as a ready-to-use assay?

Probes can be deliverd in any format, concentration or combination. Have a look at our oligo dispensing service for more information about this topic.

All your DNA oligos are delivered with a specification sheet including GC-content, molecular weight, melting temperature (Tm), OD value, sequence and quantity in μgram and nmol. 

A stringent quality control system ensures that you can expect the quality of our oligonucleotides to be of the highest standard. Before the synthesis, all chemicals are checked to meet our quality standards. During the fully automated oligo synthesis, all steps of the synthesis are monitored by multiple control functions on our DNA synthesizers. This way we can assure the coupling efficiency of the synthesis to meet our demands.

Synthesis is followed by further quality controls to guarantee the quality of the oligos. All oligos are routinely analyzed by optical density (OD) measurement. In addition, oligos are randomly analyzed by gel electrophoresis and Liquid Chromatography Mass Spectrometry (LCMS). If an oligo does not meet our requirement it’s resynthesized. The result of our stringent QC process is a product of the highest standard. All oligos are delivered with our product specification sheet, which includes: % GC content, yield in nmol and μgram, OD, melting temperature and molecular weight (MW).

  

ISO quality management

ISO 9001             

 

ISO is an independent, non-governmental membership organisation and the worlds largest developer of voluntary international standards. Biolegio’s manufacturing facility is ISO 9001:2008 and ISO 13485:2003 certified. Achieving these certificates ensures good, reliable and safe products as a result of strong product quality and manufacturing procedures and an ongoing development of our quality management system. This ongoing development empowers us to continuously strengthen our ability to identify and exceed the needs and expectations of our customers.