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Stock No: 008448
Protocol 17845: Standard PCR Assay - Rag2<tm1.1Cgn> Alternate 1
Version 3.0
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 = 156 bp
Heterozygote = 156 bp and 234 bp
Wild type = 234 bp
JAX Protocol
Protocol Primers
| Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
|---|---|---|---|---|---|---|
| 25602 | ATC AAT GGT TCA CCC CTT TG | Wild type Forward | A | |||
| 25603 | TCA TGT GAA AGC AGT TCA GGA C | Wild type Reverse | A | |||
| 25604 | CCG CCA TAT GCA TCC AAC | Mutant Reverse | A | |||
| oIMR8330 | CAG CGC TCC TCC TGA TAC TC | 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 |
| 25602 | 0.50 uM |
| 25603 | 0.50 uM |
| 25604 | 0.50 uM |
| oIMR8330 | 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 |
|---|---|
| 025730 | B6.129S-Rag2tm1Fwa Cd47tm1Fpl Il2rgtm1Wjl/J |
| 003174 | B6.129S4-Il2rgtm1Wjl/J |
| 030957 | B6.Cg-Rag2tm1.1Cgn TcrbLn4Sdou TcraLn2Sdou/J |
| 030958 | B6.Cg-Rag2tm1.1Cgn TcrbLn5Sdou TcraLn3Sdou/J |
| 008449 | B6.Cg-Rag2tm1.1Cgn/J |
| 008448 | C.B6(Cg)-Rag2tm1.1Cgn/J |
| 6 strains use this protocol | |