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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
>chr11:99540354-99540473 120bp ATCTCAGGGAGCCCACAAC TCTCTTGGTTAGGTCTCTTCTCA
Mut= 117 bp
Wt= 120 bp
Fam=Mut
Hex=Wt
Sequence
Wt Sequence (deletion in lower case):
GACATTTAAACCTAATGGAAAGAGGGGATGGGAAAATGACTCAGCAATTAAGAGTGCCGATTGTTCTTCCAAAGGACCCAAGTTCAGTTTCCCGTACCCATCTCAGGGAGCCCACAACTGTCCATAACTgtagctccgtgtggcatacaggcacacttaaatgtaaatttttaaaatctttcttaaacagaagtaatgagaagagacctaaccaagagaatctgaacttttacccagtgtgccaaaaatcaaagtgcaatgcttttttcatcacattaacatcacagacacacacacacagagcactctgacattcttcactccatttccacccacaggaggtcaacctgcttcgtgagcagcttggcgatcggctcagcgtggagctggacactgcccccactgtggacctcaacaaggtcctggatgagatgcgatgtcaatacgagagggtgctggccaacaaccgcagggatgcagaggagtggttcgctgctcaggtcagtgccctcggtaaaggactggcttccgggagggtgccctgcctgagctctctaatggggcctttgtttcagacagaagagctgaaccagcagcagaagtccagcgcggagcagttggagggctgccagactgagatgttggagttgaagcgcaaggccaacactctggaaattgagctccaggcgcagcaaaccctggtgcgtgggatgaacagtgccagcctttgtcagcagcatccagagatgctatgctctttgacagaagtccagggagatgctcacagttcagcagcaggtctgggtatccaattagcctagctggcatttccctccctttcttcacatatgttacttgtccacagtagaaacagaatgacagatcaacgggctttgtcctctcaccagggtgcctccctggccctcctaagcctaagcatgtctccaataccatcagctagagacaagaaaagtctcagaacacagtgggacagggagagctgagccaaaggcccttaagcttagcaacagattttcctttcctctaatcgatgaacagttttacagattcaccgtagtgtctaaaacaaaaaaacaaacaaacaaacaaaaaaaaaccacatcaagtttcaagtggtcatacaacagctaagacccgcacatcaccatagccctccccacccacacaagaacaccatgcagtgaaagtcacgtataccaggtcccatcaggcactgtggtaccagttgagcttcatccaaggagtttacctagatgaacgtcacctgagcatccacctaggagaggcatggtgcaatctatggcagacaccttcactagcccgggtgaacaggtgggggaaagctagggtcaaaggaagaaagttccagaatgtgctgcagttctcagtagaaacagaatcctcctggtggttgaaccctgatcttctttgtaccaacactcactgcgcagatgctcagactcaagggtttcttcacaaatgatgacaaccctccttgagctacccaaaaaaagttctggctgacaattgttctaatgatagaagcgacacatttaaaggccttgagttgcttctaggcactgttccaaattctccccaagtatcagctcatctcacctccccagcctcccccatgacataggtactttgcattctccattgactctagaataaaacaggctcagagaagctaagtaactggccctgaggtctctgagctagaaaggggtacatcgagggtctgtcatagggtccttctgagaaggttctactcttgcagacggagtctctggagtgtactgtggcagaaaccgaggcccagtacagcacacaactggcccaaatgcagtgcctaattgacagtgtggagcaccagctggctgagatccgctgtgacctggagcgtcagaaccaggagtaccaggtgctgctggacaccaaggcccggctggagtgtgagatcaacacgtacaggggcctgctggagaaggaggacagcaggcaagttacacagacccccagagtccccccaggagaaccctgtttcctcaaggcctgactaaggccaattccatgtttcggggtcttccttttcaaacagtgaaagaaaggcgtggccagGTAGCTCAGTGGGTATGTCAGTTACTTCTTTGCTTCTGTAAACATGGCCAAAGACAACTTGAGGAAGAGAGAGTTTTGCAGATGGGTAAGAGTATGTCATGGCTGCAAGCATCAGGCCTGACAGCAGGAAGT
This mutation is a 2013 bp deletion beginning at Chromosome 11 position 99,538,431 bp and ending after 99,540,443 bp (GRCm38/mm10).
JAX Protocol
Protocol Primers
| Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
|---|---|---|---|---|---|---|
| 43401 | ATC TCA GGG AGC CCA CAA C | Common | A | |||
| 43402 | TCT CTT GGT TAG GTC TCT TCT CA | Wild type Reverse | A | |||
| 43403 | ACC CAT CTG CAA AAC TCT CTC | Mutant Reverse | A | |||
| 43404 | Fluorophore-1 | AGC TCC GTG TGG CAT ACA G | Quencher-1 | WT Probe | ||
| 43405 | Fluorophore-2 | CTG TAG CTC AGT GGG TAT GTC AGT | Quencher-2 | MUT Probe |
Reaction A
| Component | Final Concentration |
|---|---|
| Kapa Probe Fast QPCR | 1.00 X |
| ddH2O | |
| 43401 | 0.40 uM |
| 43402 | 0.40 uM |
| 43403 | 0.40 uM |
| Wt Probe | 0.15 uM |
| Mutant Probe | 0.15 uM |
| 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.