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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
>chr1:161031484-161031574 91bp TCAGTGAATCAGAATAGCATCG AGACCCCTGACAATCAGCTC
Mut= 91 bp
Wt= 91 bp
Fam=Mut
Hex=Wt
Sequence
Wt Sequence (deletion in lower case):
AATACCTTGTACCAGGCTCGGCAGCTTACAGTAATCCTAGCACTTAGGAGGTAGGATCAGGAGTTTGAGGCCAGCCTTATTACTAACAAAACACCTGCTGGCAATTCTGTTTCTACATCCAAACATAAGGAAGATCTCTCTTTCTATATGAAGAATTCTTTTTTTCTCCCTGAGACAAGATTTCACTTATTTAGCCCATACTCATGATTCTCCTGCCTCTTCGGTTTTCCAAGCGCTAAAATTACGTATGTGTAGCTGGCACTCATGGAAAGAttttccatttgtagggatgtttgaactaaaaagcacaaagggagtcaaactagtactaagactgtgctttgctctttcagcagggttgaatgtctgcctgtgctttcaggcatgaccatggagaaaagtgggaacatacaattggagatccccgacttcagcaactctgttctgagccatctcaaccagctacgaatgcaaggccggctctgtgatattgtggtcaatgtgcaaggacaagcttttcgggctcacaaagtagtactggctgccagctccccttatttccgggatcacatgtccttgaatgaaatgagtactgtgtccatttctgtcatcaagaaccctactgtttttgagcaacttctttctttctgttacacagggcggatatgcctgcaactagcagatatcatcagctacctaacagctgctagttttctgcagatgcagcatattatagacaaatgtacgcagatcctagagggcattcacttcaaaattaatgtggctgaggttgaagcagaattaagtcaaacaaggacaaagcatcaagagagacctccagagtctcatagggctacaccaaatctaaaccgctcacttagtccacggcataatgcctcaaagggaaactggagagggcaggtcagtgccgtgctggatatccgagagctcagtcccccggaggagcccaccagtcctcagataattgaacagagttctgatgcagaaggccgggagcccattcttcgaatcaacagagcaggacagtggtatgtggaaacaggactagcagaccagggggcccggagtggtgacgaagttagggttcttggagcagtgcacatcaaaactgaaaatctagaagagtggcttgggccagagaatcagccttctggagaagatgggagtagtgcagaggaagtcacagccatggtgattgacaccacaggccatggttctataggacaggaaagttatactttagggtcttcaggagccaaggtagctcggccaaccagcagtgaagttgacaggtaagttttgtttctccttttaaagctattgtacagacatcattaaataacctttctgtgttacagagggcatctttttttacctgatattcttattcagcttactttgggagactggaagtatgtcagaagacttgcattttcagcacaggaggctcctggatctgaaatattgacgatgttatgagaactgtggtaaactgtggtaaactgtggtaaaattagccacttcagtgaatcagaatagcatcggcagccactctaggatcagtaattgccagggctctcTGCACTAGGCAGGGAGCTGATTGTCAGGGGTCTGGTAACATTGCTGTTTCTCACACAGGTAGGTAACCTTGCGGAGTCTGACTTGCCTTATAGCACTGTGTATTTCTCTTTGCCCTTACCGACAGAGACAATTTGGTACCATCAATAATTTTCAAATTAGAGTAATTACACTACAGTAATTCTAGAGAAAAGCTTTTGAATGTTTTCCAAGTGTCACCTGGTAATGTTTCCTTTGAAGCAGCTACTACCCCAGGCCTGTTTCTTCCTAACATACTGAGTCTG
This mutation is a 1328 bp deletion beginning at Chromosome 1 position 161,031,517 bp and ending after 161,032,844 bp (GRCm38/mm10).
JAX Protocol
Protocol Primers
| Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
|---|---|---|---|---|---|---|
| 48635 | TCA GTG AAT CAG AAT AGC ATC G | Wild type Forward | A | |||
| 48636 | AGA CCC CTG ACA ATC AGC TC | Common | A | |||
| 48637 | CCT CTT CGG TTT TCC AAG C | Mutant Forward | A | |||
| 48638 | Fluorophore-1 | CTA GGA TCA GTA ATT GCC AGG G | Quencher-1 | WT Probe | ||
| 48639 | Fluorophore-2 | CGT ATG TGT AGC TGG CAC TCA T | Quencher-2 | MUT Probe |
Reaction A
| Component | Final Concentration |
|---|---|
| Kapa Probe Fast QPCR | 1.00 X |
| ddH2O | |
| 48635 | 0.40 uM |
| 48636 | 0.40 uM |
| 48637 | 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.