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Stock No: 007908
Protocol 29436: Standard PCR Assay - Gt(ROSA)26Sor(tdTomato-WPRE)
Version 2.2
Notes
Melt curve analysis is done using a Roche Light Cycler 480.
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 Tm = 81°C +/- 1.0°C
Wild type Tm = 87°C +/- 1.0°C
Mutant = 196 bp
Heterozygote = 297 bp and 196 bp
Wild type = 297 bp
For Stock# 012567
Mutant = ~315 bp
Wild type = 297 bp
For Stock #7914
Mutant = ~200 bp
Wild type = 297 bp
JAX Protocol
Protocol Primers
| Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
|---|---|---|---|---|---|---|
| oIMR9020 | AAG GGA GCT GCA GTG GAG TA | Wild type Forward | A | |||
| oIMR9021 | CCG AAA ATC TGT GGG AAG TC | Wild type Reverse | A | |||
| oIMR9103 | GGC ATT AAA GCA GCG TAT CC | Mutant Reverse | A | WPRE | ||
| oIMR9105 | CTG TTC CTG TAC GGC ATG G | Mutant Forward | A | tdTomato |
Reaction A
| Component | Final Concentration |
|---|---|
| ddH2O | |
| Kapa 2G HS buffer | 1.30 X |
| MgCl2 | 2.60 mM |
| dNTP KAPA | 0.26 mM |
| oIMR9020 | 0.50 uM |
| oIMR9021 | 0.50 uM |
| oIMR9103 | 0.50 uM |
| oIMR9105 | 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 |
|---|---|
| 031800 | B6.Cg-Gad2tm1Rpa Gad1tm1Rpa Gt(ROSA)26Sortm14(CAG-tdTomato)Hze/RpaJ |
| 038839 | B6.Cg-Gt(ROSA)26Sortm1.1(CAG-Venus,-Sdk2)Jrs/KrishJ |
| 007914 | B6.Cg-Gt(ROSA)26Sortm14(CAG-tdTomato)Hze/J |
| 012567 | B6.Cg-Gt(ROSA)26Sortm27.1(CAG-COP4*H134R/tdTomato)Hze/J |
| 032864 | B6.Cg-Gt(ROSA)26Sortm65.2(CAG-tdTomato)Hze/J |
| 025106 | B6.Cg-Gt(ROSA)26Sortm75.1(CAG-tdTomato*)Hze/J |
| 007909 | B6.Cg-Gt(ROSA)26Sortm9(CAG-tdTomato)Hze/J |
| 008538 | B6.Cg-Tg(Cspg4-cre/Esr1*)BAkik/J |
| 008863 | B6.Cg-Tg(Tek-cre)1Ywa/J |
| 012570 | B6;129S-Gt(ROSA)26Sortm34.1(CAG-Syp/tdTomato)Hze/J |
| 021875 | B6;129S-Gt(ROSA)26Sortm65.1(CAG-tdTomato)Hze/J |
| 021876 | B6;129S-Gt(ROSA)26Sortm66.1(CAG-tdTomato)Hze/J |
| 007908 | B6;129S6-Gt(ROSA)26Sortm14(CAG-tdTomato)Hze/J |
| 007905 | B6;129S6-Gt(ROSA)26Sortm9(CAG-tdTomato)Hze/J |
| 030855 | STOCK Notch1tm2Rko Notch2tm3Grid Gt(ROSA)26Sortm9(CAG-tdTomato)Hze Tg(KRT5-cre/ERT2)2Ipc/JeswJ |
| 15 strains use this protocol | |