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Notes
To run this assay on agarose you will need to run two separate reactions; one for the wild type Rag1 allele (Wild type forward and common primers) and one for the Rag1 knockout allele (mutant forward and common primers).
The genotyping protocol(s) presented here have been optimized for reagents and conditions used by The Jackson Laboratory (JAX).
To genotype animals, JAX recommends researchers validate the assay independently upon receipt of animals into their facility.
Reaction cycling temperature and times may require additional optimization based on the specific genotyping reagents used.
Expected Results
Mutant = 197 bp
Heterozygote = 197 bp and 192 bp
Wild type = 192 bp
If running on a gel run separated:
23267and 23268 for Wt
23268 and oIMR8162 for Mut
JAX Protocol
Protocol Primers
| Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
|---|---|---|---|---|---|---|
| 23267 | TCT GGA CTT GCC TCC TCT GT | Wild type Forward | A | |||
| 23268 | CAT TCC ATC GCA AGA CTC CT | Common | A | |||
| oIMR8162 | TGG ATG TGG AAT GTG TGC GAG | Mutant Forward | A | PGK R |
Reaction A
| Component | Final Concentration |
|---|---|
| ddH2O | |
| Kapa 2G HS buffer | 1.30 X |
| MgCl2 | 2.60 mM |
| dNTP KAPA | 0.26 mM |
| 23267 | 0.50 uM |
| 23268 | 0.50 uM |
| oIMR8162 | 0.50 uM |
| Glycerol | 6.50 % |
| Dye | 1.00 X |
| Kapa 2G HS taq polym | 0.03 U/ul |
| DNA |
Cycling
| Step | Temp °C | Time | Note |
|---|---|---|---|
| 1 | 94.0 | -- | |
| 2 | 94.0 | -- | |
| 3 | 65.0 | -- | -0.5 C per cycle decrease |
| 4 | 68.0 | -- | |
| 5 | -- | repeat steps 2-4 for 10 cycles (Touchdown) | |
| 6 | 94.0 | -- | |
| 7 | 60.0 | -- | |
| 8 | 72.0 | -- | |
| 9 | -- | repeat steps 6-8 for 28 cycles | |
| 10 | 72.0 | -- | |
| 11 | 10.0 | -- | hold |
JAX uses a very high speed Taq (~1000 bp/sec), use cycling times recommended for your reagents.
JAX uses a 'touchdown' cycling protocol and therefore has not calculated the optimal annealing temperature for each set of primers.
Strains Using This Protocol
| Stock Number | Strain Name |
|---|---|
| 017700 | B6.129-Rag1tm1Mom Fcgrttm1Dcr/DcrJ |
| 002216 | B6.129S7-Rag1tm1Mom/J |
| 016919 | B6.Cg-Rag1tm1Mom Fcgrttm1Dcr Tg(CAG-FCGRT)276Dcr/DcrJ |
| 004369 | B6.Cg-Rag1tm1Mom Ins2Akita/J |
| 008684 | B6.Cg-Rag1tm1Mom Tyrp1B-w Tg(Tcra,Tcrb)9Rest/J |
| 013115 | B6.Cg-Rag1tm1Mom Tg(UBC-GFP)30Scha/J |
| 002096 | B6;129S7-Rag1tm1Mom/J |
| 021571 | BXSB.129S7(B6)-Rag1tm1Mom/DcrJ |
| 003145 | C.129S7(B6)-Rag1tm1Mom/J |
| 030666 | C.Cg-Rag1tm1Mom Tg(DO11.10)10Dlo/J |
| 003729 | NOD.129S7(B6)-Rag1tm1Mom/J |
| 004347 | NOD.Cg-Rag1tm1Mom Tg(TcraAI4)1Dvs/DvsJ |
| 004348 | NOD.Cg-Rag1tm1Mom Tg(TcrbAI4)1Dvs/DvsJ |
| 030442 | NOD.Cg-Rag1tm1Mom Dmdmdx-4Cv Il2rgtm1Wjl/McalJ |
| 026062 | NOD.Cg-Rag1tm1Mom Fancaem1Dvs Il2rgtm1Wjl/SzJ |
| 006022 | NOD.Cg-Rag1tm1Mom H2-Ab1b-tm1Gru Tg(CD2-CD4,HLA-DQA1,HLA-DQB1)1Ell/EllJ |
| 006024 | NOD.Cg-Rag1tm1Mom H2-Ab1b-tm1Gru Tg(CD4,HLA-DQA1,HLA-DQB1)N8Ell/EllJ |
| 008659 | NOD.Cg-Rag1tm1Mom Ins2Akita Prf1tm1Sdz/SzJ |
| 026014 | NOD.Cg-Rag1tm1Mom KitW-41J Il2rgtm1Wjl/EavJ |
| 004848 | NOD.Cg-Rag1tm1Mom Prf1tm1Sdz/SzJ |
| 030443 | NOD.Cg-Rag1tm1Mom Sgcatm1Kcam Il2rgtm1Wjl/McalJ |
| 029663 | NOD.Cg-Rag1tm1Mom Dysfprmd Il2rgtm1Wjl/McalJ |
| 024099 | NOD.Cg-Rag1tm1Mom Il2rgtm1Wjl Tg(CMV-IL3,CSF2,KITLG)1Eav/J |
| 017914 | NOD.Cg-Rag1tm1Mom Il2rgtm1Wjl Tg(HLA-DRA,HLA-DRB1*0401)39-2Kito/ScasJ |
| 007799 | NOD.Cg-Rag1tm1Mom Il2rgtm1Wjl/SzJ |
| 014568 | NOD.Cg-Rag1tm1Mom Ins2Akita Il2rgtm1Wjl/SzJ |
| 009377 | NOD.Cg-Rag1tm1Mom Tg(TcraBDC12-4.1)10Jos Tg(TcrbBDC12-4.1)82Gse/J |
| 005707 | STOCK Rag1tm1Mom Tg(TIE2-lacZ)182Sato/J |
| 006770 | STOCK Rag1tm1Mom Tg(TIE2GFP)287Sato/J |
| 29 strains use this protocol | |