FAQs

Oligonucleotides

General

• Does my oligonucleotide have a phosphate on the 5' or the 3' end?
• G-rich oligonucleotides, can they reliably be synthesized?
• How should I store my oligo and how long will it last?
• How do I calculate the molecular weight of an oligonucleotide?
• What are the IUB codes for mixed bases?

Oligo quantification

• How does Syntezza quantify its oligos?
• What does 1 OD stand for?
• To estimate how much of a freeze-dried oligonucleotide I have, should I rely on the weight in mgs or the absorbance (O.D. value)?

Real-Time PCR

General information on Real-Time PCR kits

• What is the advantage of working with a probe system?
  
• How should the qPCR and RT qPCR kits be stored?
  
• What is the difference between a Core kit and a MasterMix?
  
• What is the difference between a MasterMix and a MasterMix Plus?
  
• Should the DNA and the qPCR MasterMix be mixed before thermocycling?
  
• What is the difference between a One step and a Two step RT qPCR reaction?
  
• In which case is one step RT qPCR recommended?
  
• Why is a Two step RT qPCR kit more efficient than a One step RT qPCR kit?
  
• Why do the qPCR and RT qPCR kits contain HotGoldStar, a hotstart Taq polymerase?
  
• Why do the Syntezza RT qPCR kits contain EuroScript, a MmuLV reverse transcriptase?
  
• Why do qPCR kits contain dUTP (and UNG)?
  
• Why does an initial step at 50°C during 2 min and a second step at 95°C during 10 min have to be performed when using UNG for carry-over prevention?
  
• Why do the one step RT qPCR MasterMixes not contain UNG?
  
• Why do certain qPCR kits contain ROX passive reference?
  
• Can an "ABI" kit with a ROX passive reference be used on an IQ i-cycler?
  
• What should I do to use a ROX or a No ROX qPCR Core kit or MasterMix for SYBR^® green I correctly on an IQ i-cycler?
  
• How do I avoid amplification of genomic DNA, which can be a contamination factor in my isolated RNA in RT qPCR?

Theory behind Real-Time PCR

• What can cause a SYBR^® green I reaction, which has been working well, stop working?
• How to optimize a qPCR assay?
• In which concentration should I use my probes in qPCR?
• In which concentration should I use my primers in qPCR?
• Are DNA or RNA standards preferred in qPCR?
• What is UNG and how does it work?
• Is there a difference in PCR efficiency when using dUTP instead of dTTP?
• How to select a good endogenous control?
• What is the difference between endpoint analysis and Real-Time analysis?
• How can the perfomance of allelic discimination be increased?
• What is absolute quantitation?
• What is relative quantitation?

Probes

• In which buffer to resuspend qPCR probes?
• How should the qPCR probes be stored?
• How many reactions can be done with one batch of Double-Dye oligo?
• What can cause a very high background level when working with probes?
• How can a high background level be avoided when working with TAMRA, ROX, Cy^®3 or Cy^®5 as fluorophore in a Double-Dye oligo?
• How can a low signal to noise ratio for probes be explained?
• How can a low signal to noise ratio for Molecular Beacons be explained?
• Which probe systems can be used for expression profiling?
  
• Which probe systems can be used for allelic discrimination?
• How do typical cycling conditions for a Double-Dye oligo on a 96-well Real-Time thermocycler look like?
• Which fluorescent dye should I use for my target and controls?
• Which dyes are preferably used on Double-Dye oligos?
• Which fluorophores can be combined in a multiplex Double-Dye oligo assay?
• Can a FAM-TAMRA Double-Dye oligo be combined with a Yakima Yellow™-Dark Quencher Double-Dye oligo?
• Can Yakima Yellow™, as fluorophore, be combined with TAMRA, as quencher, on a Double-Dye oligo?
• Can Yakima Yellow™ used with VIC settings?
• Should anything be changed in the software when analyzing double dye oligos with quenchers other than TAMRA?
• Does Syntezza do design for Real-Time probes?

No template control is positive

• What can cause No Template Controls to give a positive result?
• What can cause No Template Controls to give a positive result in the presence of UNG?
• How can negative control be positive when detecting E. Coli sequences?

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