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Stock No: 026179
Protocol 21168: Standard PCR Assay - Generic Rosa<#m1.1(CAG-CAS9)>
Version 2.0
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 (28555, 28551) (Rsky) = 172 bp
Mutant (30936, 26179 and 24858) (Fezh, Dvs, & Mvw) = 290 bp
Wild type = 109 bp
JAX Protocol
Protocol Primers
| Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
|---|---|---|---|---|---|---|
| 21306 | CTG GCT TCT GAG GAC CG | Wild type Forward | A | |||
| 24493 | AGC CTG CCC AGA AGA CTC C | Wild type Reverse | A | |||
| 26932 | CTC CGT CGT GGT CCT TAT AGT | Mutant Reverse | A | |||
| oIMR5984 | GCT AAC CAT GTT CAT GCC TTC | Mutant Forward | A |
Reaction A
| Component | Final Concentration |
|---|---|
| ddH2O | |
| Kapa 2G HS buffer | 1.30 X |
| MgCl2 | 2.60 mM |
| dNTP KAPA | 0.26 mM |
| 21306 | 0.50 uM |
| 24493 | 0.50 uM |
| 26932 | 0.50 uM |
| oIMR5984 | 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 |
|---|---|
| 028555 | B6(C)-Gt(ROSA)26Sorem1.1(CAG-cas9*,-EGFP)Rsky/J |
| 026179 | B6J.129(Cg)-Gt(ROSA)26Sortm1.1(CAG-cas9*,-EGFP)Fezh/J |
| 026430 | B6N.129(Cg)-Gt(ROSA)26Sortm1.1(CAG-cas9*,-EGFP)Fezh/J |
| 028551 | C57BL/6N-Gt(ROSA)26Sorem1(CAG-cas9*,-EGFP)Rsky/J |
| 026558 | FVB.129(B6)-Gt(ROSA)26Sortm1.1(CAG-cas9*,-EGFP)Fezh/J |
| 026557 | NOD.129(Cg)-Gt(ROSA)26Sortm1.1(CAG-cas9*,-EGFP)Fezh/J |
| 030936 | NOD/ShiLtJ-Gt(ROSA)26Sorem1.1(CAG-cas9*,-EGFP)Dvs/J |
| 024858 | STOCK Gt(ROSA)26Sortm1.1(CAG-cas9*,-EGFP)Fezh/J |
| 8 strains use this protocol | |