My PCR won't work. What should I do?
PCR protocols developed in one lab may not always work well under other lab conditions. Troubleshoot and optimize using the content and links provided here. Consider ordering mice that have already been genotyped for your experiments, or having a vendor perform your genotyping for you. Although The Jackson Laboratory does not provide genotyping as a service, there are vendors that perform this service that can be found in online searches.
How can I tell the difference between a homozygous Tg/Tg mouse and a hemizygous Tg/0 mouse?
Homozygous and hemizygous transgenic mice can be distinguished using one of three techniques:
- Southern blot. Reference: Reiter, A. et al. 1997. Quantitative Southern blot for monitoring CML patients on therapy. British Journal of Haematology 97: 86.
- Quantitative PCR (qPCR): See references: Liu et al. 2003. Quantitative PCR genotyping assay for the Ts65DN mouse model of Down syndrome. BioTechniques 35: 1-7;
Tesson L et al. 2002. Rapid and accurate determination of zygosity in transgenic animals by real-time quantitative PCR. Transgenic Res. 11(1):43-8.
Shitara et al. 2004. Simple method of zygosity identification in transgenic mice by real-time quantitative PCR. Transgenic Res. 13(2):191-4.
- Progeny testing: Mate mice that test positive for the transgene (Tg/?) to wild-type mice and genotype the offspring. If any of the offspring fail to carry the transgene, the Tg/? parent is hemizygous Tg/0. If all the offspring test positive for the transgene (10-12 offspring should be tested), the Tg/? parent is homozygous Tg/Tg.
What is an internal control?
Internal control primers typically amplify a genomic DNA region that is unrelated to your gene of interest. Successful amplification of the internal control indicates your DNA is suitable for PCR.
Why is SYBR green used in some PCR assays?
SYBR green is commonly used in melting curve analysis, a PCR method that determines band sizes without an agarose gel. You can modify a SYBR green assay for standard PCR by eliminating the SYBR green and optimizing the protocol for your own conditions using a standard thermocycler.
What is melting curve analysis?
The melting point of double-stranded PCR products can be used to determine the size of the DNA fragments amplified (eliminating the need for agarose gels). Melting curve protocols are performed with standard PCR reagents but require a fluorescent dye that binds double-stranded DNA (typically SYBR green), and use of a special thermocycler. Melting curve analysis protocols provided on JAX® Mice data sheets can be used as a starting point for standard PCR, by eliminating the SYBR green and optimizing the protocol for use in a standard thermocycler.
How can I determine where the primers are binding?
The Basic Local Alignment Search Tool (BLAST) database can be used to identify where primers bind in the genome. Review of the primary reference from the donating investigator who developed the mouse strain may also provide information on primer binding sites. See the “References” tab on the strain data sheets for some key references.
Do you have other primers or other protocols available for JAX® Mice?
Everything available is displayed on the strain data sheets. If you need additional help, the primary reference may provide alternative primers or genotyping protocol to the one posted on our website. See the “References” tab on the strain data sheet.
Do you have construct information used to develop the mouse so I can develop new primers?
No. Information on the genetic constructs is only available through the primary reference(s) published by the investigator who donated the strain to The Jackson Laboratory. Key references can be found on the “References” tab for each strain data sheet in the JAX® Mice Database.
How do I calculate the melting temperature (Tm) for new primers?
Melting temperatures can be calculated with the formula Tm = 2[A+T] + 4[G+C], or calculated online.
There was no protocol available online for my strain. What do I do?
Genotyping protocols are not available for all JAX® Mice strains. You can check the primary reference(s) from the investigator who donated the strain or develop your own assay based on information found in the literature.
What if the JAX® Mice protocol provided doesn't work in my lab?
PCR protocols developed in one lab don't always work under different lab conditions. Troubleshoot and optimize using the content and links provided here or consider having a vendor perform your genotyping for you. Although The Jackson Laboratory does not provide genotyping as a service, there are vendors that perform this service that can be found in online searches.
What is pyrosequencing?
Pyrosequencing is a DNA-sequencing method that uses a chemiluminescent enzyme to detect specific nucleotide incorporation into DNA.