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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
>chr4:155840675+155840758 84bp GGGTAACATGGCAACAAACC AACGCTGTAGGTATTTTTCACC
Mut= 81 bp
Wt= 84 bp
Fam=Mut
Hex=Wt
Sequence
Wt Sequence (deletion in lower case):
CCTGCAGAGAGTCACATCCTTTCCTACTTCTCGCTGCCAGATGTCTGGTTTCCCACTCACTCTCCCAGAACTTGTTAAGTGGGTAACATGGCAACAAACCTGCCATGACTGCAGGAGGCCCcaagtggtcctggggtgcaaaggtgaaaaatacctacagcgttattcctcctcttcttccaggctctgcagtcctgtcctcaggtcagtatcccctatccagctgtgggccagaccccttcagggtccactcctgtcctagtcctgcctcctctcttgacttctgccactgactcccccttcccgacaagggccttcagggaccgcagcagccagcagctctctggtgtctgagtctgtggtgagcttggcagccggaacccaggctgtgctacgctgccagagcccccgcatggtgtggacccaagaccggctgcatgatcgccagcgcgtggtccactgggacctcagcgggggcccgggcagccaacggcgccgacttgtggatatgtattcggcgggtgaacagcgcgtgtacgagccgcgcgatcgcgaccgcctcctgctgtcgccttctgctttccacgacggcaacttctcgctgctcattcgcggtacactgtagaaccgtagtgggctgaaactttggcagaaactgaaaccttggcggcaggggtgagggtggggtggaggagtggggaggtgtggtgggtagggggagggagctgagaggccttcctaaggttggagccttggatgggcggggccctagaagggattggccgattttaggggaggggccatagagaggacgggttccctcaatctaagggtatcttctgccaagcagctgtggacagaggcgatgaaggggtgtacacctgcaacctgcaccatcactactgccacctcgatgagagcctggctgtgcgcctcgaggttacagaggatcgtgagtgtgtcccttcctctccttcgctatttccatccctatcctccttacccccaatcttccccctcctcacctctgcgaggtgatctgactgcctttctcaacgcatccgcagccctattaagtcgcgcatactgggacggtgagaaggaagtgttggtggtggcccatggcgcgccggcactgatgacctgcatcaaccgtgcgcacgtgtggactgaccgccatttagaggaggcgcagcaggtggtccattgggaccgacagctacctggggtgtcacacgaccgcgccgaccgcctgcttgacctgtatgcatctggcgagcgccgcgcctatgggccacccttcctgcgtgatcgcgtgtcagtgaacaccaacgcttttgcacgcggtgacttctccctacgcatcgatgagctggagcgagctgatgagggcatctattcctgccacctgcaccatcactactgtggcctccacgagcgccgagtcttccacctacaggtcacagagcctgcctttgagccaccagctcgtgcttctcctggcaatgggtctggtcacagcagtgctcctagcccaggtgtgtgtgtagcctgccggcctgagaagaggtcctgttgtgacctagattgctaattgctagaggtattgccacggctgcctgcctgcctgcctgcctgccttccttccttccttccttccttccttccttccttccttccttccttccttccttccttccttccttcctttcctttccttccttccttccttccttccttcttttccttttcctcctcctcttcctccttccttctcctcctttttttttttttggcttttgtttttttgagacagggtttcttctaggctgtcctggaaccaggctgggctcaaactcaccgagatctgtctgcctctgcctcctgagtgatgagattaaaggcatgtaccaccatcacctggcttgcagaggttttctgatgcttgctacttacatctagatcccaccctgacccgaggccacagcatcatcaatgtcattgtcccagaggaccacacacatttcttccagcaactgggctatgtgttggccacgctgctgctcttcatcttgctgctcatcactgtagtcctggctacacgatatcgtcacagcggaggtgaggctgggcccttggaacagaggggcccagattaaccgagaacagtgagagcaacccaataactctcttcacaggatgcaagacgtcggacaaaaaagctgggaagtcaaaggggtgagtttgcctgcccaactctcctcccagacctgtcctgccctggcctcctaaactgctatctccctttaggaaggatgtgaacatggtggagtttgctgtagccacaagggatcaggctccatataggactgaggacatccagctaggtaggggtgagagccacaagggtccaggggaagtacgttttggagtggcacttattattatttttttccccaaccagattacaaaaacaacatcctgaaggagagggctgagctggcccatagtcctctgcctgccaaggatgtggatctggataaaggtgagcacttttacctcctcatcccatatgagactctacccattccagggactccctgccattttcctgagaaggtcttgggaagtcatgccaaatgtctccattgactttcccactgggactaagggacagagagcagacattagccattttcttggcttccagagttcaggaaggagtactgcaaataaatggaccctgagcttctggctgggccagcagctctgtatcaaaggacatctccctgaccctcctgcggtattcctggctcttctcagcggctggtccgacttacctagaaacttggcagagcagctgcctgtactttgcccttcctagaatcgccacccctcatcttggtgagcaactgtgggttccctagagactctggtatagtacgattgctgcccttcacctgtgcccactgatggttgtacccccaacttaaacacaacaaagatcccttgttaatatccaccaaatgcaaagtccctcgtggcctcttactgctagggtcaggaagacacttaaaaattccagttaagactccctagccaccagttaaacacattagccattgtcctggggggtcttcctgagctgcattgtgcctgtgtactgttcagagccctgctgttataggttctgactcatgggcccgccttgctgctttgggcaacttgaggctagcccagggccctttctctgcttctgattcctttctgccgaatgcctcccaagagctacaccagcagttactgggtaccgtatgactcttggccttgacatccctccctaggctggagtctggggttggggccccatttgtcctctgttttggctgaagatggggcgaagatttggctgagtggcctatgctgtcacatcaaacagctatcatttactcctacttgggaagttttcatgtgacaataaaagatacatctgatttttagtcttggacctgctgctctccttctgttcaggaaggccaagtctgggttttgggaacctccagctgctcccataacctgatccccttgggggaaggtctacgtttgtgtccttctagggtcagatgtcctatgacacaggaacctgttttccatggtgaagggctttcagctccttttttgaagtgtgtgtgtgtgtgtgtgtgtgtgtagggagggggtggtatacaatggtgGGAGGGAGGTTGGCCCTAGAAGGAAATGCTGTTAGCCCAATTCTTTGAGAAGCAAATGCTGAGATGAAGTAAAAGGAAGAAGCAAAGATAGGCAGGCATGTTCCCCAAGGATACAAATCTGAAGA
This mutation is a 3613 bp deletion beginning at Chromosome 4 position 155,840,716 bp and ending after 155,844,328 bp (GRCm38/mm10).
JAX Protocol
Protocol Primers
| Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
|---|---|---|---|---|---|---|
| 42891 | GGG TAA CAT GGC AAC AAA CC | Common | A | |||
| 42892 | AAC GCT GTA GGT ATT TTT CAC C | Wild type Reverse | A | |||
| 42893 | TTG GGC TAA CAG CAT TTC CT | Mutant Reverse | A | |||
| 42894 | Fluorophore-1 | TGG TCC TGG GGT GCA A | Quencher-1 | WT Probe | ||
| 42895 | Fluorophore-2 | AGG GAG GTT GGC CCT AGA | Quencher-2 | MUT Probe |
Reaction A
| Component | Final Concentration |
|---|---|
| Kapa Probe Fast QPCR | 1.00 X |
| ddH2O | |
| 42891 | 0.40 uM |
| 42892 | 0.40 uM |
| 42893 | 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.