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Notes
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
Mutation = 350 bp
Transgene = 420 bp
Internal Positive Control = 200 bp
XX female= 200 bp
XY- female= 350 bp and 200 bp
XX male= 420 bp and 200 bp
XY- male = 420 bp and 350 bp and 200 bp
Transgene = 420 bp
Internal Positive Control = 200 bp
XX female= 200 bp
XY- female= 350 bp and 200 bp
XX male= 420 bp and 200 bp
XY- male = 420 bp and 350 bp and 200 bp
JAX Protocol
Protocol Primers
| Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
|---|---|---|---|---|---|---|
| 9369 | CTG GAG CTC TAC AGT GAT GA | Forward | ||||
| 9370 | CAG TTA CCA ATC AAC ACA TCA C | Reverse | ||||
| 9371 | AGC CCT ACA GCC ACA TGA TA | Transgene Forward | Sry | |||
| 9372 | GTC TTG CCT GTA TGT GAT GG | Transgene Reverse | Sry | |||
| oIMR8744 | CAA ATG TTG CTT GTC TGG TG | Internal Positive Control | ||||
| oIMR8745 | GTC AGT CGA GTG CAC AGT TT | Internal Positive Control |
Reaction A
| Component | Final Concentration |
|---|---|
| ddH2O | |
| KAPA Rxn Buffer Contains 1.5 mM Mg2+ final concentration. | 1.00 |
| MgCl2 | 0.00 |
| dNTP | 0.20 |
| 9369 | 1.00 |
| 9370 | 1.00 |
| 9371 | 1.00 |
| 9372 | 1.00 |
| oIMR8744 | 0.10 |
| oIMR8745 | 0.10 |
| DNA Loading Dye | 0.69 |
| KAPATaq DNA Polymera No proofreading function. 1 error per 40000 nucleotides. Initial denature 2 min. | 0.03 |
| DNA |
Cycling
| Step | Temp °C | Time | Note |
|---|---|---|---|
| 1 | 94.0 | -- | |
| 2 | 94.0 | -- | |
| 3 | 60.0 | -- | |
| 4 | 72.0 | -- | |
| 5 | -- | Go to step 2-4, 35 times | |
| 6 | 72.0 | -- | |
| 7 | 10.0 | -- | hold |
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 |
|---|---|
| 010905 | B6.Cg-Tg(Sry)2Ei Srydl1Rlb T(XTmsb4x-Hccs;Y)1Dto/ArnoJ |
| 039108 | B6.Cg-Tg(Sry)2Ei Srydl1Rlb/JtuJ |
| 002063 | C57BL/6JEi-Tg(Sry)1Ei Chr YAKR/J/EiJ |
| 002708 | C57BL/6JEi-Tg(Sry)2Ei Chr YAKR/J/EiJ |
| 002837 | C57BL/6JEi-Tg(Sry)3Ei Chr YAKR/J/EiJ |
| 002183 | C57BL/6JEi-Tg(Sry)4Ei Chr YPOS/EiJ |
| 027001 | QSi5/IanmTaftJ |
| 7 strains use this protocol | |