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FAQ - Primer Pairs and Standards

Q: What are SybGREEN qPCR Primer Pairs and panels?
A: SybGREEN qPCR Primer Pairs are pre-designed, qPCR tested and ready-to-use primer sets for SYBR Green based qPCR experiments. The primer pairs are also available in a 96-well panel format covering broad range of pathways for cancer biomarker profiling.

Q: Can SybGREEN qPCR Primer Pairs be used in probe-based qPCR?
A: No. This kit has not been designed to accommodate any commercial probe based qPCR.

Q: What is the Tm of the primer and what is a typical size of the amplicon?
A: The Tm of a SybGREEN qPCR Primer is around 60, and the amplicon is around 95 to 140 bp.

Q: Does a SybGREEN qPCR Primer Pair amplify an exon junction in a cDNA target?
A: Whenever possible, an amplicon by a SybGREEN qPCR Primer Pair covers exon junction or junctions.

Q: My qPCR machine is not on the list of compatible machines on the website. Do you provide qPCR primer panels in plates compatible with other machines?
A: Yes, we can provide our primer panels in other plates within approximately one week of receiving the custom order. Please email the catalog numbers of the qPCR primer panels needed plus the catalog number and manufacturer of the plates to techsupport@origene.com. Our technical support scientists will prepare a custom quote that can be used to order custom panels.

Q: If I provide a list of genes I want to assay, can OriGene prepare a custom qPCR primer panel?
A: Yes, OriGene can design a custom qPCR primer panel. Custom panels are offered for human and mouse genes in 200 reactions format for purchase, delivered in 2-D bar code matrix tubes.

Q: Where can I find the sequence of the primers in each panel?
A: Each panel is delivered with a USB drive that contains a spreadsheet of primer sequences and locations.

Q: How much primer pair is in each well?
A: Each well in 200 reactions format contains 2 nmol of lyophilized primer pair. Please re-dissolve in 200ul of water so the final concentration is 10uM. Each well in the PCR plate contains 10pmol of primer pairs. The primers will be re-suspended once the PCR reagents are added to the plate.

Q: What is a qPCR template standard?
A: A gene specific qPCR template standard is a tube of linear DNA made from a full-length cDNA plasmid. The copy number of a template standard solution is determined by the PicoGreen method and calculation based on MW.

Q: Can a template standard be used in a probe-based qPCR?
A: Yes, as long as a corresponding probe is used.

Q: In your protocol, it is recommended to make six serial dilutions with one log of difference, can I make more dilutions and with different dilution scheme?
A: Yes, as long as it is diluted in the qPCR detection range and each dilution is mixed thoroughly.

Q: Can I use my own buffer to dilute the template standard instead of the buffer in the kit?
A: Yes, as long as the buffer has no negative effects on qPCR.

Q: Does OriGene offer custom-made template standards?
A: Yes. Please contact our Tech Support at techsupport@origene.com

Q: What is the OriGene guarantee on qPCR primers and template standards?
A: OriGene qPCR primers and templates are warranted for SYBR Green qPCR experiments. If your experimental results are not satisfactory, our scientists will work with you to pinpoint the problem and, if it is determined that our products are at fault, OriGene will refund your money in the form of a credit.

Q: How should I calculate the copy number of my gene of interest?

  1. If the cDNA templates in your samples are single-stranded such as cDNA from RT reactions, theactual copy number of your gene of interest is two times the number you got by comparing to theqPCR standards. For example, if the copy number of your gene is 5 copy/μl from the standardcurve of a qPCR program, the actual number is 10 copy/μl. The reason is that the first cycle for asingle-stranded sample is to make the complementary strand; therefore, there is one cycle delayin the PCR reaction compared to the double-stranded cDNA template standard's reaction.
  2. If the templates in your samples are double-stranded, the copy number of your gene of interest isthe same as that calculated according to the qPCR standards.

Q: I am writing a paper for publication and need to describe this product. How should I cite?
A: We recommend that you refer to the product by its specific catalog number and refer to us as OriGene Technologies (Rockville, MD). Furthermore, we'd love to hear from you when your paper is published. Inform us and we will send a gift.


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