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Stock No: 032864
Protocol 35289: Separated PCR Assay - Gt(ROSA)26Sor<tm65.2(CAG-tdTomato)Hze>
Version 1.0
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 = 290 bp
Heterozygote = 290 bp and 402 bp
Wild type = 402 bp
>chr6:113025997-113026398 402bp ACGGGCAGTAGGGCTGAG AGCCTGCCCAGAAGACTCC
JAX Protocol
Protocol Primers
| Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
|---|---|---|---|---|---|---|
| 24493 | AGC CTG CCC AGA AGA CTC C | Wild type Reverse | A | |||
| 44393 | ACG GGC AGT AGG GCT GAG | Wild type Forward | A | |||
| 45697 | TCT AGC TTG GGC TGC AGG T | Mutant Reverse | B | |||
| 45698 | GGT GTT GGG TCG TTT GTT CA | Mutant Forward | B |
Reaction A
| Component | Final Concentration |
|---|---|
| ddH2O | |
| Kapa 2G HS buffer | 1.30 X |
| MgCl2 | 2.60 mM |
| dNTPS-kapa | 0.26 mM |
| 24493 | 0.50 uM |
| 44393 | 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.
Reaction B
| Component | Final Concentration |
|---|---|
| ddH2O | |
| Kapa 2G HS buffer | 1.30 X |
| MgCl2 | 2.60 mM |
| dNTPS-kapa | 0.26 mM |
| 45697 | 0.50 uM |
| 45698 | 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 |
|---|---|
| 032864 | B6.Cg-Gt(ROSA)26Sortm65.2(CAG-tdTomato)Hze/J |
| 038629 | STOCK Gt(ROSA)26Sortm65.2(CAG-tdTomato)Hze Pdgfrbtm1.1(flpo)Leol/LeolJ |
| 038627 | STOCK Pdgfratm1(flpo)Leol Gt(ROSA)26Sortm65.2(CAG-tdTomato)Hze/LeolJ |
| 038628 | STOCK Pdgfratm2.1(flpo)Leol Gt(ROSA)26Sortm65.2(CAG-tdTomato)Hze/LeolJ |
| 4 strains use this protocol | |