The Jax recommended PCR primers never worked well for us.  The following protocol is quite robust in our hands, even for crude ProK DNA preps from ear punches.  This is easier than bleeding mice for Va2/Vb5 staining by FACS (which is also a reliable  approach for identifying animals expressing the OTII TCR):

Valpha2 (B22) 5’-TGGCCAGCAGAAGGAGAA-3’  

Calpha (B23) 5'- GGTGAAGCTTGTCTGGTT-3’ 

Reaction Mix (Make master mix on ice; aliqot into PCR tubes/plate on ice; add DNA last)

*[DNA  1.0 uL]

10X PCR buffer 2 uL

2mM dNTPs 2 uL

10uM B22 1.6 uL

10 uM B23 1.6 uL

Taq 0.2 uL

H20 11.6 uL

[Total 20.0 uL]

PCR

(Preheat lid. Assemble tubes on ice. Quickly transfer tubes to heat block when thermocycler block reaches ~85 C, for a "hot start" effect)

35 cycles using the following program:

95 C, 20 s

54 C, 20 s

72 C, 45 s

Hold at 4 C

Analysis of Products

Run on a 1.2-1.5% agarose gel. Desired product is 580bp.

__________

*The crude ear punch DNA prep we use is as follows:

1. Obtain ear punch or tail snip and place in eppendorf tube. 

Add 150 l ear buffer: 10 mM Tris, pH 8.0, 50 mM KCl, 0.5% NP40, 0.5% Tween

3. Heat to 95 C (not higher) for 10 minutes.

4. Add 3 uL proteinase K (stock: 10 mg/mL in 10 mM Tris, pH 8.0 – works best if you make fresh; frozen stock can also be used, but loses activity over a period of several weeks).

5. Incubate at 56 C for 3 hours. Vortex briefly every hour.

6. Heat to 95 C (not higher) for 15 minutes to inactivate Proteinase K.

7. Quick spin, vortex, and quick spin again.

7. Use 1-2 l in PCR reaction (in a 20 uL total volume)

8. Samples can be stored at -20 C and re-used/re-tested several times.

What you propose is phenotyping, not genotyping. Genotyping must be based on genomic DNA extracted from your mice and is the only safe way of knowing what are the genotypes of your animals. You can proceed either by standard PCR or qPCR. If you use standard PCR, you should design a set of PCR oligos that detect your wild-type TCR locus so you can make the difference between homo- and heterozygous mice. It is not very clear what type of Ly5.1 mice you're using are they Cre or Tg over-expressing mice? I have no experience with your strains, but you should be very careful and refer to the original publications to set up a clean PCR genotyping protocol. I have not experience with qPCR genotyping either, but is looks a bit difficult, I expect just one Ct difference between Hetero and Homo, apparently there is only one probe against Tg allele. In case of qPCR genotyping, I would advise (but I might be wrong) probes against both mutant and wild-type alleles and multiplexing (costly!).

With all the best wishes from a serial genotyper how's got his lot of genotyping "surprises" (like matted "homozygous" mutant mice giving birth to wild-types!).

The Jax recommended PCR primers never worked well for us.  The following protocol is quite robust in our hands, even for crude ProK DNA preps from ear punches.  This is easier than bleeding mice for Va2/Vb5 staining by FACS (which is also a reliable  approach for identifying animals expressing the OTII TCR):

Valpha2 (B22) 5’-TGGCCAGCAGAAGGAGAA-3’  

Calpha (B23) 5'- GGTGAAGCTTGTCTGGTT-3’ 

Reaction Mix (Make master mix on ice; aliqot into PCR tubes/plate on ice; add DNA last)

*[DNA  1.0 uL]

10X PCR buffer 2 uL

2mM dNTPs 2 uL

10uM B22 1.6 uL

10 uM B23 1.6 uL

Taq 0.2 uL

H20 11.6 uL

[Total 20.0 uL]

PCR

(Preheat lid. Assemble tubes on ice. Quickly transfer tubes to heat block when thermocycler block reaches ~85 C, for a "hot start" effect)

35 cycles using the following program:

95 C, 20 s

54 C, 20 s

72 C, 45 s

Hold at 4 C

Analysis of Products

Run on a 1.2-1.5% agarose gel. Desired product is 580bp.

__________

*The crude ear punch DNA prep we use is as follows:

1. Obtain ear punch or tail snip and place in eppendorf tube. 

Add 150 l ear buffer: 10 mM Tris, pH 8.0, 50 mM KCl, 0.5% NP40, 0.5% Tween

3. Heat to 95 C (not higher) for 10 minutes.

4. Add 3 uL proteinase K (stock: 10 mg/mL in 10 mM Tris, pH 8.0 – works best if you make fresh; frozen stock can also be used, but loses activity over a period of several weeks).

5. Incubate at 56 C for 3 hours. Vortex briefly every hour.

6. Heat to 95 C (not higher) for 15 minutes to inactivate Proteinase K.

7. Quick spin, vortex, and quick spin again.

7. Use 1-2 l in PCR reaction (in a 20 uL total volume)

8. Samples can be stored at -20 C and re-used/re-tested several times.

Thanks a lot to you all, this was really helpful!

I don't know if you guys still follow this thread, but I found the discussion useful as I'm just starting a small OT-II colony in our lab, and I was hoping to optimize the genotyping PCR. I tried the primers suggested by Dr Schaefer and I got 2 major bands in an animal that I believe is heterozygous for the OT-II transgene (founder male purchased from Taconic). The brighter one was at the suggested ~580bp and the other one was slightly below 400bp. I assume the 580bp is the band for the transgene, but I was wondering if the other one was a non-specific band or an endogeneous "wildtype" TCR band? Does your PCR distinguish between heterozygous and homozygous animals? Thanks!!!

Hi Carla,

With transgenic mice produced by DNA microinjection and random integration of transgenes (such as OTII), the precise integration site is almost never known. Without knowing the sequence surrounding the integration site, there is no way to design primers for the "WT" allele (or more specifically, the native chromosomal region with no integrated transgene). With the right strategy and high-quality DNA preps, it should be possible to design real-time PCR primers for the OTII transgene sequence to discriminate heterozygotes and homozygotes (i.e., 2-fold higher abundance for homozygotes), but we have not done this.

Hope this helps.