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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
>chr2:52416793-52416912 120bp CTGCCTGGAGAGATTGTTGG GCCTTCCTAGAGCTCAAAAGATAC
Mutant= 137 bp
Wild Type = 120 bp
Sequence
Wt sequence with deletion in lower case
TTCAGTAACTTAAGTCATTTGTAACTGTTGTGACTACATTTGAGGATCCTTTGCTGAGCATAGACTTCCTTAGGCCTAGAAAGAGAAGGTGCTTCTGACTGCCTGGAGAGATTGTTGGAAAGATGAGAAGTGGCCGTGTAGaatgagaagaggcaaagaaagtgagcttaacaacacagtccttgcttgatggagtatcttttgagctctaggaaggctaaagagtgttgtgtgctgacttataccacagaaacaaagaagaataaagtgtgggatctctgtgtgtggtaactctctgctggtttttatttcagagcacaaagttatcattgttgggctggacaatgctgggaagacgaccatcctttaccagttgtaagtattaactgcttaaagtttaattttgttgtttctagttattctctgtgcttttgtggtatttgatggatgaaatcaatgaaaagttatgtggatgcaagcagactaaccgtaagccagaagccagggactgtgtgctgagctagaaattcgcacgataccaggcatgctcattgttttgctttgatcctcttgtttttagaaaacaaggtatctgtttatacactttgcctgaatgatctgtaatattctaaaaccattatttatttgagctcttttatatctttagctgaatcattgtgtgagcgcaaaaacaagggcaattgggtctgagttagaagccatgttgacgtaccttgatgtaagcattggtgagatgaactagcttcctcactaattttgtaaacttttcccctcctagttccatgaatgaagtcgtacacacatctcctacaataggaagcaatgtggaagagattgtggttaataatacccgtttcctgatgtgggatattggtggccaagaatcacttcgtccttcctggaacacgtactatactaacacagaggtaacattcctaatgctagtgcttgcatgagctcctgagtcctggaacacgtactatactaacacagaggtaacattcctaatgctagtgcttgcatgagctcctgagtccttgagaagcattgggttcgagtcattacagtctttaaaaatggagcgtgagtacagtagtcttctattccagtcttgactcagttaacgttgagtctctgtagGGAGAGTTGGTGGGTGATCAGTCGGTTGTTTGTCCAGTAGTTACTCTTAAATGACTTGCATTTCATGATTTCTGATTGCATTTCCTTCTTGATGTTTATATGTGTCTGAGTGGGAGAAATCCCAGCCAAGTAACGGACTCTTATGTGGTTTGAGTCCTAACCAGTTTTTGCCGTGTATTGTCTTTTATGTAGTGTAGTTGCAGCTGAAAAATCTTTTAATCATAAAGCATTTTACACATAATCATATGGTTTTATTTACATGTTACATTC
A 1020 bp deletion beginning at Chromosome 2 position 52,415,850 bp and ending after 52,416,869 bp (GRCm38/mm10).
JAX Protocol
Protocol Primers
| Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
|---|---|---|---|---|---|---|
| 45112 | CTG CCT GGA GAG ATT GTT GG | Common | A | |||
| 45113 | GCC TTC CTA GAG CTC AAA AGA TAC | Wild type Reverse | A | |||
| 45114 | CAT CAA GAA GGA AAT GCA ATC A | Mutant Reverse | A | |||
| 45115 | Fluorophore-1 | AGC TTA ACA ACA CAG TCC TTG CTT | Quencher-1 | WT Probe | ||
| 45116 | Fluorophore-2 | TCG GTT GTT TGT CCA GTA GTT ACT C | Quencher-2 | MUT Probe |
Reaction A
| Component | Final Concentration |
|---|---|
| Kapa Probe Fast QPCR | 1.00 X |
| ddH2O | |
| 45112 | 0.40 uM |
| 45113 | 0.40 uM |
| 45114 | 0.40 uM |
| Wt Probe | 0.15 uM |
| Mutant Probe | 0.15 uM |
| DNA |
Cycling
| Step | Temp °C | Time | Note |
|---|---|---|---|
| 1 | 95.0 | -- | |
| 2 | 95.0 | -- | |
| 3 | 60.0 | -- | |
| 4 | -- | repeat steps 2-3 for 40 cycles | |
| 5 | 40.0 | -- | Forever |
JAX uses a very high speed Taq (~1000 bp/sec), use cycling times recommended for your reagents.
Strains Using This Protocol
This is the only strain that uses this protocol.