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Notes
Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.
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
Sequence
Wt Sequence (deletions in lower case):
CACTAGTCTCGTGCCCAGCATCTCTCCTCCCCACGCACCGACAACCCTGGGCCGCcgcggggcgggggatgccgggctgccgaatcagcgcctgcggcccaggggcccaggaagggacggcagaaccggggtcgcccccgccgccaccccgggagcccctgccgtccctccagcccccttccccatcgccgacctcgactccgacccccactaagtcaccgcccttacccgaggcggccgagacacccgtggaagggcaggagctgcagcgctggcgccagggtgctagtggggggtctgggggcgcgggcccggcggggatcgcgggcgcggcggcgggggcagggggccgtgcgctggagctggccgaagcgcggcggcgactgctagaagtggagggccgccggcgcctggtgtcggagctggagagccgggtgctgcagctgcaccgagtcttcttggctgcggagctgcgcctggcgcatcgcgccgagagcctgagccgcctgagcggcggcgtggcccaggccgagctctacctggctgcgcacgggtcccgtctcaagaagggtgcgcgccgcggtcgccggggtcgtcccccggcgctgctggcgtccgcgctcggcctggggagctgcgtgccctggggcgcagggcggctacggcgcggccagggcccggagcccgactcgcccttccgccgcAGCCCGCCCCGCGGCCCCGCCTCCCCCCAGCGCTGACCTCAGTACCCCGGACCCCTGGCTTCCCGAGGCAGGCGAGTCTCCAGGTGCGGGAGGAC
This mutation is a 649 bp deletion beginning at Chromosome 4 position 133,497,822 bp and ending after 133,498,470 bp (GRCm38/mm10).
JAX Protocol
Protocol Primers
| Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
|---|---|---|---|---|---|---|
| 51541 | AGT CTC GTG CCC AGC ATC TC | Common | A, B | |||
| 51542 | TTC TGC CGT CCC TTC CTG | Wild type Reverse | A | |||
| 51543 | CTC CCG CAC CTG GAG ACT | Mutant Reverse | B |
Reaction A
| Component | Final Concentration |
|---|---|
| ddH2O | |
| Kapa 2G HS buffer | 1.30 X |
| MgCl2 | 2.60 mM |
| dNTPS-kapa | 0.26 mM |
| 51541 | 0.50 uM |
| 51542 | 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 |
| 51541 | 0.50 uM |
| 51543 | 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
This is the only strain that uses this protocol.