For in-depth product & services help, ask our
Technical Information Scientists
Stock No: 002292
Protocol 27172: Standard PCR Assay - Gt(ROSA)26Sor STD
Version 2.2
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
HET = 374 bp and ~550 bp
MUT = ~550 bp
WT = 374 bp
Separated by gel electrophoresis on a 1.5% agarose gel.
JAX Protocol
Protocol Primers
| Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
|---|---|---|---|---|---|---|
| 20483 | GGC TTA AAG GCT AAC CTG GTG TG | Wild type Forward | A | |||
| oIMR0092 | AAT CCA TCT TGT TCA ATG GCC GAT C | Mutant Reverse | A | Neo R | ||
| oIMR3449 | CCG GAT TGA TGG TAG TGG TC | Mutant Forward | A | LacZ F | ||
| oIMR8546 | GGA GCG GGA GAA ATG GAT ATG | Wild type Reverse | A |
Reaction A
| Component | Final Concentration |
|---|---|
| ddH2O | |
| Kapa 2G HS buffer | 1.30 X |
| MgCl2 | 2.60 mM |
| dNTP KAPA | 0.26 mM |
| 20483 | 0.50 uM |
| oIMR0092 | 0.50 uM |
| oIMR3449 | 0.50 uM |
| oIMR8546 | 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 |
|---|---|
| 002292 | 129-Gt(ROSA)26Sor/J |
| 002192 | B6.129S7-Gt(ROSA)26Sor/J |
| 003504 | B6;129-Gt(ROSA)26Sortm1Sho/J |
| 002955 | C.129S7-Gt(ROSA)26Sor/J |
| 029855 | STOCK Sncatm1.1Vlb Gt(ROSA)26Sortm1Sho/J |
| 028560 | STOCK Sncatm1.2Vlb Gt(ROSA)26Sortm1Sho/J |
| 6 strains use this protocol | |