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FAQ - Primer Pairs and Standards
Q: What are SybGREEN qPCR Primer Pairs?
A: SybGREEN qPCR Primer Pairs are pre-designed, qPCR tested and ready-to-use primer sets for SYBR Green based qPCR experiments.
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: 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 credit
Q: How should I calculate the copy number of my gene of interest?
A:
- If the cDNA templates in your samples are single-stranded such as cDNA from RT reactions, the actual copy number of your gene of interest is two times the number you got by comparing to the qPCR standards. For example, if the copy number of your gene is 5 copy/µl from the standard curve of a qPCR program, the actual number is 10 copy/µl. The reason is that the first cycle for a single-stranded sample is to make the complementary strand; therefore, there is one cycle delay in the PCR reaction compared to the double-stranded cDNA template standard’s reaction.
- If the templates in your samples are double-stranded, the copy number of your gene of interest is the same as that calculated according to the qPCR standards.
Q: I need to cite your product for a paper I am writing. What language should I use?
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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