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Notes
TCAAACTATTATCTTAAGCAAGTAGCTGTGGATATAAACTATGTCTCATTAATTTTATTTTAATACTTACCACAGCAATACCTCAAGATACCAAAACACATGGCCAACTGTGGGAATATTCTGATAGAAGAGAATATTATTGGGTAGTCTTCAGATCAGTGGGATAACTAGCCAAAAAGAATCTATGAAAGAAATCTAATCTAAATGATCCAGGATAGGTCTAAGAACTTCACTGAGATTTTATTAATTCACAATTAATCAAAAGATAGGTATTCATGATTCAAGCTGGCTATGTAAATACTTCTTTTCAGTGTCTTATTTTTCCCATTATAACTTTTTCTGTTTTTAACAGTTCCAGAAACGCCTAAGCCTAGTTGTGGGGATCCAGCTGCAAGAAAAACGTTAGTCTCTTGGCCTGAGCCTG(T/C)ATCTAAACCTGA(G/T)(T/C)CTGCATCTAAACCCCATGGATATGTTTTATGCTATAAGAACAATTCAGGTAATGTAAAATTCCACTAGGGAAACAAAATCAAGAATTTTTAAATGTTGTAATTATTGTTTTAGCCAGGGAGCTACAAGGCTTTTATGAAGGTGATGAGAGATTCATGTTTGAGTTTGCTTAGATGAACACGCATCTCATGGAGAAGAATAAATATCTAGATCATTTATTAAAAATAGTGGACAGGACTAGAGTCCTTTAGAGCAGGTTACTCATCAGGACATTCCTTTATCTCTGGGCTAAGGTGGGAATGTACTTAGCTCTCATAGA
Twelve nucleotide differences between the a and b alleles have been identified. This assay is designed to detect a T to C point mutation (indicated in red), however, additional nucleotide differences in close proximity have been indicated in green.
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 (Ptprc<a>)(CD45.1, Ly5.1) = C/C
Heterozygote = T/C
Wild type (Ptprc<b>)(CD45.2, Ly5.2) = T/T
JAX Protocol
Protocol Primers
| Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
|---|---|---|---|---|---|---|
| 15560 | CGC CTA AGC CTA GTT GTG G | Forward | A | |||
| 15561 | ATT CTT GAT TTT GTT TCC CTA GTG G | Reverse | A | |||
| 25560 | Fluorophore-1 | CCT GAG CCT GTA TCT AAA CCT GA | Quencher-1 | WT Probe | ||
| 25561 | Fluorophore-2 | CCT GAG CCT GCA TCT AAA CCT G | Quencher-2 | MUT Probe |
Reaction A
| Component | Final Concentration |
|---|---|
| Kapa Probe Fast QPCR | 1.00 X |
| ddH2O | |
| 15560 | 0.40 uM |
| 15561 | 0.40 uM |
| Wt Probe | 0.15 uM |
| Mutant Probe | 0.15 uM |
| DNA |
Cycling
| Step | Temp °C | Time | Note |
|---|---|---|---|
| 1 | 95.0 | -- | |
| 2 | 95.0 | -- | |
| 3 | 60.0 | -- | repeat steps 2-3 for 40 cycles |
JAX uses a very high speed Taq (~1000 bp/sec), use cycling times recommended for your reagents.
Strains Using This Protocol
| Stock Number | Strain Name |
|---|---|
| 007594 | B6.129P2-Ptprca Ightm1Mnz/J |
| 028269 | B6.129S6(C)-Inpp5dtm1.1Wgk/J |
| 006183 | B6.Cg-Col4a5tm1Yseg/J |
| 006259 | B6.Cg-Pepcb Ptprca Tg(UBC-scFv)2Nemz/J |
| 040031 | B6.Cg-Ptprca Tg(Rorc-tdTomato)1Goet/J |
| 008451 | B6.Cg-Ptprca Cx3cr1tm1Litt/LittJ |
| 013198 | B6.Cg-Ptprca Mir223tm1Fcam/J |
| 037061 | B6.Cg-Ptprca Pepcb Tg(EEF1A1-IL15RA)1Nsat/J |
| 030450 | B6.Cg-Ptprca Pepcb Tg(TcrLCMV)1Aox/PpmJ |
| 039112 | B6.Cg-Ptprca Pepcb KitW-41J Gpi1a/IslabMmjax |
| 027509 | B6.Cg-Ptprca Pepcb Tg(TcraBC10,TcrbBC10)3Chi/J |
| 022486 | B6.Cg-Ptprca Tg(UBC-PA-GFP)1Mnz/J |
| 002014 | B6.SJL-Ptprca Pepcb/BoyJ |
| 007203 | B6;129S4-Zfand5Gt(ROSA)72Sor/J |
| 025854 | B6;FVB-Ptprca Tg(CAG-luc,-GFP)L2G85Chco Thy1a/J |
| 017351 | BKa.Cg-Ptprcb Bmi1tm1Ilw Thy1a/J |
| 007686 | BKa.Cg-Sox17tm2Sjm Ptprcb Thy1a/J |
| 018869 | C.129S4(Cg)-Il13tm2.1Lky/J |
| 006584 | CByJ.SJL(B6)-Ptprca/J |
| 035855 | NOD.Cg-Rag1tm1Mom Il2rgtm1Wjl Tg(HLA-A/H2-D/B2M)Dvs Tg(HLA-DRA,HLA-DRB1*0401)39-2Kito/J |
| 025855 | STOCK Ptprca Lag3tm1Doi Tg(CAG-luc,-GFP)L2G85Chco Thy1a/J |
| 037371 | STOCK Ptprca Pepcb H2u H2-T18a Tg(CD2-TcraAc1-9,-TcrbAc1-9)4Kio/WdcJ |
| 030113 | STOCK Ptprca Pepcb Pkmtm1.1Mgvh/J |
| 028062 | STOCK Ptprca TcrbLn1Bdb TcraLn1Bdb H2d/J |
| 24 strains use this protocol | |