- Where do I find the genotyping protocol for my strain?
- I don’t see a standard genotyping assay. Do you have one for my strain?
- Why isn’t the protocol on your website working? Why aren’t these primers working?
- Do I have to use the same genotyping reagents that are listed in your protocols?
- What is the KAPA2G taq that you refer to in the protocols?
- How do you isolate DNA?
- I think the mice I received are the wrong genotype. What should I do?
- What is a probe assay/an endpoint assay/melt curve analysis/touchdown PCR?
- How can I tell the difference between a homozygous Tg/Tg mouse and a hemizygous Tg/0 mouse?
- Do you have construct information used to develop the mouse so I can develop new primers?
- I see online that the genotyping assay I’ve been using has been updated. Why did you do that? What has changedWhere has the old one gone? Can I get the old one? Is there a problem with the old one?
- What is an internal control?
- Why is SYBR green used in some PCR assays?
- What is melting curve analysis?
- How can I determine where the primers are binding?
- Do you have other primers or other protocols available for JAX® Mice?
- Do you have construct or sequence information used to develop the mouse so I can develop new primers?
- How do I calculate the melting temperature (Tm) for new primers?
- There was no protocol available online for my strain. What do I do?
- What is pyrosequencing?
A. Genotyping FAQ
1. Where do I find the genotyping protocol for my strain?
- Look in the Technical Support section of the strain datasheet for linked protocols
- Click here to access the database where you can enter your strain’s stock #
- Alternate protocols may be available in the published literature. We often are not provided genotyping protocols for excised alleles (a floxed allele after a cross to cre). This information can often be found in the primary (or another) reference for a strain.
2. I don’t see a standard genotyping assay. Do you have one for my strain?
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.
3. Why isn’t the protocol on your website working? Why aren’t these primers working?
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:
- We use a very fast Taq polymerase. It may be helpful to increase the length of the elongation steps in the cycling protocol (usually steps 4 & 8) to match your taq (a standard taq can polymerize 1000 bp/min).
- Many customers get better results when they run reactions separately, not as a multiplex. This means running two reactions for each sample.
- Use proper controls. It is difficult to troubleshoot when controls are not being run. Appropriate controls may include the following:
- Known positive for the mutant alleles
- Known wild-type or non-carrier (negative for the mutant alleles)
- No template control (primers only)
- An internal control primer set, which will clarify that the absence of a transgene band is not due to poor DNA quality
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.
4. Do I have to use the same genotyping reagents that are listed in your protocols?
No, the protocols can work with other reagents. You may need to optimize the conditions for the assay to work in your lab.
5. What is the KAPA2G taq that you refer to in the protocols?
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.
6. How do you isolate DNA?
Please read our PCR Tips (Section B)
7. I think the mice I received are the wrong genotype. What should I do?
You will need to provide genotyping data showing the error, including the use of proper controls.
- Known positive for the mutant alleles
- Known wild-type or non-carrier (negative for the mutant alleles)
- No template control (primers only)
- If relevant, an internal control primer set, which will clarify that the absence of a transgene band is not due to poor DNA quality
8. What is a probe assay/an endpoint assay/melt curve analysis/touchdown PCR?
Please read our blog article: Hold the Agarose! Advanced PCR Methods for Genotyping Mice
9. 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. Reiter, A. et al. 1997. Quantitative Southern blot for monitoring CML patients on therapy. British Journal of Haematology 97: 86
- Quantitative PCR (qPCR):
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.
10. 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 on each strain datasheet.
11. I see online that the genotyping assay I’ve been using has been updated. Why did you do that? What has changed?
Where has the old one gone? Can I get the old one? Is there a problem with the old one?
Sometimes we need to optimize assays to fit our high-throughput needs. The changes are usually minor and typically the old assay works fine.
12. 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.
13. 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.
14. 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.
15. 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.
16. 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 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.
17. Do you have construct or sequence 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.
18. 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.
19. 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.
20. What is pyrosequencing?
Pyrosequencing is a DNA-sequencing method that uses a chemiluminescent enzyme to detect specific nucleotide incorporation into DNA.
B. PCR Tips
General Tips when collecting tissues (tail tip or ear punch), clean tools with 70% ETOH in between animals.
- Use filtered pipette tips.
- Avoid repeated freezing and thawing of nucleotides. Aliquot small volumes and store at -70°C.
- Do NOT perform PCR in a ventilated hood as it increases the risk of cross-contamination.
- Mix the reaction tube by gentle tapping. Do not vortex PCR mix.
- Add DNA polymerase (Taq) to the reaction tube last.
- Adjust electrophoresis voltage and run time to improve band resolution.
- Confirm that the PCR machine was programmed correctly.
- Avoid overloading PCR products into the gel; this may result in cross-contamination or misinterpretation of the results.
Genotyping protocols for JAX® Mice are provided as a starting point for developing your own assay.
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.
Alter the amount of DNA per reaction.
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.
Test 'Touchdown cycling' as an alternative for multiplex PCR reactions.
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 multiplexing a reaction is ineffective, separate primer pairs.
If one PCR product is weak or absent in a multiplex reaction, run separate PCR reactions for each primer pair and optimize them independently.
Dilute fresh PCR reagents and/or perform a new DNA isolation/dilution.
If there are unexpected changes in your PCR results (especially in controls) or if contamination is suspected, obtain freshly-made reagents.
Use a different DNA isolation protocol.
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).
Double-check primer sequences if control bands are absent.
If the sequence is correct, consider ordering new primers if degradation or hidden production errors could be the problem.
Consider designing new primers or a new PCR assay.
Genotyping protocols for JAX® Mice are provided as a starting point to develop your own assay. References by the primary investigators who donated the strain typically contain the most extensive genetic information needed to develop new primers and protocols. Primary references are found on all JAX® Mice Database strain data sheets.
Increase or decrease the annealing temperature.
If the expected bands are not amplified, dropping the annealing temperature can help primers bind more efficiently. If bands are weak, try using a longer extension time.
If there are more bands than expected (non-specific bands are amplified), raising the annealing temperature can help eliminate non-specific primer binding.
Try a 'Hot start' to reduce non-specific bands.
Pre-heat the thermocycler to 94°C before adding samples, or use a hot-start-specific Taq polymerase (available from most companies that sell Taq), or use a Taq polymerase antibody in the reaction mixture according to manufactures instructions (i.e. TaqStart, BD Biosciences/Clontech, stock# 639250). A 1:1 TaqStart antibody to Taq DNA Polymerse ratio is commonly used for a hot-start procedure.
Use Perfect Match® PCR Enhancer to increase specificity.
This reagent destabilizes mismatched primer-template complexes to reduce PCR artifacts, thereby promoting amplification of the correct PCR products.
To reduce false positives in neo genotyping assays, use new reagents and clean equipment (filtered pipette tips, clean pipettes, a clean lab bench space etc.).
Neo contamination is common and can lead to false positives when genotyping mice using neo-specific primers. You can also develop a mutation-specific PCR assay that uses mutation-specific (rather than neo-specific) primers.
C. DNA Isolation Protocols
Quick DNA purification protocol
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
- Cut 2mm of tail and place into an Eppendorf tube or 96-well plate.
- Add 75ul 25mM NaOH / 0.2 mM EDTA.
- Place in thermocycler at 98ºC for 1 hour, then reduce the temperature to 15°C until ready to proceed to the next step.
- Add 75ul of 40 mM Tris HCl (pH 5.5).
- Centrifuge at 4000rpm for 3 minutes.
- Take an aliquot for PCR (use 2 ul undiluted, or 2 ul of a 1:100 dilution/reaction).
Phenol/chloroform extraction from mouse tail biopsies
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:
- 50 mM Tris-HCl pH 8.0
- 100 mM EDTA pH 8.0
- 100 mM NaCl
- 1% SDS
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.
DNA extraction from blood samples
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.
1. Lysis buffer
- 0.32 M sucrose
- 10mM Tris-HCl (pH 7.5)
- 5 mM MgCl2
- 1% v/v Triton X-100
2. PBND (PCR buffer with nonionic detergents)*
- 50 mM KCl
- 10 mM Tris-HCl (pH 8.3)
- 2.5 mM MgCl2
- 0.1 mg/ml gelatin
- 0.45% (v/v) Nonidet P40
- 0.45% (v/v) Tween 20
- Autoclave to sterilize and dissolve gelatin.
- Store frozen.
*Add proteinase K (60 µg/ml) immediately prior to use). (Adapted from Higuchi, R. (1989) Amplifications 2: 1-3)
D. Genotyping-Related Articles and Resources
1. Hold the Agarose! Advanced PCR Methods for Genotyping Mice
2. Designating Genotypes: What Does '+' Really Mean?
3. PCR Troubleshooting Guide (New England Biolabs)
4. Additional Genotyping Resources