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Genotyping at JAX is optimized for a high-throughput operation. Check the protocol, even if it is not labelled a “standard PCR assay”, to see if amplicon sizes are listed. If listed, then the assay can likely be used as a traditional agarose-based assay.
PCR protocols developed in one lab may not always work well under other lab conditions. Troubleshoot and optimize using the content provided here.
Three of the most common solutions for our protocols follow:
If problems persist, 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.
No, the protocols can work with other reagents. You may need to optimize the conditions for the assay to work in your lab.
KK5519- 2G Fast hot Start (All Standard PCR both Gel based and Melt)
KK4703- Probe Fast MasterMix (2X) Universal-10mL (All Endpoint and QPCR)
The specific KAPA2G product that we use is not commercially available. These are comparable to the product we use.
Please read our PCR Tips (Section B)
You will need to provide genotyping data showing the error, including the use of proper controls.
Learn more about our Credit Policy.
Please read our blog article:
Hold the Agarose! Advanced PCR Methods for Genotyping Mice
Homozygous and hemizygous transgenic mice can be distinguished using one of three techniques:
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.
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 on each strain datasheet.
Sometimes we need to optimize assays to fit our high-throughput needs. The changes are usually minor and typically the old assay works fine.
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.
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.
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.
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.
Everything available is displayed on the strain data sheets. If you need additional help, the primary reference or other references may provide alternative primers or a genotyping protocol to the one posted on our website. See the “References” tab on the strain data sheet.
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.
Melting temperatures can be calculated with the formula Tm = 2[A+T] + 4[G+C], or calculated online.
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.
Pyrosequencing is a DNA-sequencing method that uses a chemiluminescent enzyme to detect specific nucleotide incorporation into DNA.
An assay that works well in one lab may not work well in another. Conditions and components need to be optimized for your lab conditions. In some cases, you may need to optimize extensively or develop a new assay to obtain acceptable results under your lab conditions.
Too much or too little DNA will result in poor amplification. Run a dilution series of DNA samples including a non-diluted sample, and samples diluted at 1:10, 1:100, and 1:1000 with H2O. If you know the concentration of each DNA solution, a 25μl PCR reaction typically requires ~5ng of highly-purified DNA or 40-50 ng of quick prep ("dirty") DNA - see DNA Isolation Protocols (Section C) for preparation methods.
Touchdown cycling is a common feature on many thermocyclers (consult your manual for instructions). This feature alters the temperature incrementally during the annealing step to accommodate primer pairs with different optimal annealing temperatures.
If one PCR product is weak or absent in a multiplex reaction, run separate PCR reactions foreach primer pair and optimize them independently.
If there are unexpected changes in your PCR results (especially in controls) or if contamination is suspected, obtain freshly-made reagents.
For most PCR protocols, a quick, "dirty" DNA preparation is sufficient, but some assays improve when highly purified DNA is used. See DNA Isolation Protocols (Section C).
A quick "dirty" prep is usually sufficient, while some genotyping may work better with highly purified DNA. Determine empirically which protocol works best for your genotyping.
NaOH extraction (quick "dirty" DNA preparation). Reference: Truett GE et al. 2000. Biotechniques 29(1):52-54
This protocol yields a highly purified DNA preparation from mouse tail biopsies.
1. Remove 0.5 mm of tail into polypropylene microfuge tube (do not mince). (The tubes must have tight-fitting caps, so that there are no leaks in steps 3 and 7 below.)
2. Add 0.5 ml DNA digestion buffer with proteinase K added to 0.5 mg/ml final concentration. (0.5 mg/ml is a high concentration and can probably be reduced.)
DNA digestion buffer:
3. Incubate overnight at 50-55 °C with gentle shaking. (At this step, mechanical agitation greatly aids complete disruption of the tail.)
4. Quick-spin tubes to get solution off inside of cap.
5. Fill inside well of microfuge tube cap with vacuum grease. (We use Dow Corning high-vacuum grease and a 10cc syringe to dispense.)
6. Add 0.7 ml neutralized phenol/chloroform/iso-amyl alcohol (25:24:1).
7. Mix fairly vigorously. (Do NOT vortex; we use a clinical rotator for 1 hour.)
8. Spin in microfuge at top speed 5 minutes and transfer 0.5 ml of the upper phase to new microfuge tube. (Use P1000 for transfer, and draw the aqueous phase gently through tip several times after transfer if the DNA is still in large, gelatinous mass.)
9. Add 1 ml 100% ethanol at room temperature and invert (using clinical rotator if you wish) until DNA precipitate forms. (approximately 1 minute).
10. Spin in microfuge 5 minutes and carefully remove and discard supernatant.
11. Add 0.5-1 ml 70% ethanol (-20 °C) and invert several times.
12. Spin in microfuge 5 minutes and carefully remove and discard supernatant.
13. Quick-spin tubes and remove last drop of ethanol solution with 25 µl capillary tube.
14. Air dry at room temperature or in dessicator (overnight if you wish).
15. Add 100-200 µl TE buffer and incubate at 65 °C for 15 minutes to resuspend DNA. Draw DNA through P1000 tip after 65 °C incubation to aid in suspension if you wish.
16. Use 10-20 µl for restriction enzyme digest.
17. Total yield is approximately 20-50 µg DNA, 0.1-0.25 µg/µl.
1. Obtain 65-100 µl of blood by retro-orbital bleed with a heparinized microcapillary tube. Expel blood immediately into a 1.5 ml microfuge tube containing 20 µl of 10 mM EDTA. Mix immediately to prevent clot formation. Store on ice until processing.
2. Add 200 µl lysis buffer to each tube and vortex to suspend evenly.
3. Microfuge 25 seconds at 16000xg to pellet nuclei.
4. Remove and discard supernatant and repeat steps 2-4 two more times, or until no hemoglobin remains.
5. Resuspend nuclear pellet in 100 µl PBND with 60 µg/ml proteinase K and incubate at 55 C for 60 minutes (or overnight, if convenient).
6. Heat samples to 97 C for 10 minutes to inactivate proteinase K.
7. Add 1-5 µl of DNA solution for a 25 µl PCR reaction.
Reagents:
1. Lysis buffer
2. PBND (PCR buffer with nonionic detergents)*
*Add proteinase K (60 µg/ml) immediately prior to use). (Adapted from Higuchi, R. (1989) Amplifications 2: 1-3)