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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
Mutant = 273 bp
Heterozygote =273 bp and 460 bp
Wild type = 460 bp
JAX Protocol
Protocol Primers
| Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
|---|---|---|---|---|---|---|
| 12143 | CAG GAC TGA AAA GGG AAT CA | Mutant | A | |||
| 12144 | CGT TGG AAG GCC TTG AAT TT | Mutant | A | |||
| 19391 | ATG CCC TCT TCT TGA GCT TG | Wild type | A | |||
| 19392 | AGG CTT GGA GAA GGA GGA AG | Wild type | A |
Reaction A
| Component | Final Concentration |
|---|---|
| ddH2O | |
| Kapa 2G HS buffer | 1.30 X |
| MgCl2 | 2.60 mM |
| dNTP KAPA | 0.26 mM |
| 12143 | 0.50 uM |
| 12144 | 0.50 uM |
| 19391 | 0.50 uM |
| 19392 | 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 |
|---|---|
| 017712 | C;129S4-Rag2tm1.1Flv Csf1tm1(CSF1)Flv Csf2/Il3tm1.1(CSF2,IL3)Flv Thpotm1.1(TPO)Flv Il2rgtm1.1Flv Tg(SIRPA)1Flv/J |
| 017711 | C;129S4-Rag2tm1.1Flv Csf1tm1(CSF1)Flv Csf2/Il3tm1.1(CSF2,IL3)Flv Thpotm1.1(TPO)Flv Il2rgtm1.1Flv/J |
| 014594 | C;129S4-Rag2tm1.1Flv Thpotm1.1(TPO)Flv Il2rgtm1.1Flv/J |
| 3 strains use this protocol | |