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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
>chr5:136982022-136982119 98bp TCTTCTTGTAGCCTCCATTG CAGCAGCTAAGAAAGGCAGA
Mut= 116 bp
Wt= 98 bp
Fam=Mut
Hex=Wt
Sequence
Wt Sequence (deletion in lower case):
GCCCTCTCTCCAGCTGCAATTTATTGTGTCTCCAGTTATTGCTGGGACTGGGAGGACTTAGAGGCAGCATAAAGGGCCGAAACAAGCTTGCCAAGCTGGAAGAAAGCAGGGTGAAGATCTGGGAATTCCTCACAGCATAATGAAGGGGCCGAGGCGAGGAGACTGGGGCTCTAGGCAACGGCAGTGCAACTTTCCTCCTGTCTTGACCCCGgtaactctctttgccccatctagttcgctcccaggaccctggacagcggcgggtgctggacagggcagccaggcagcggcggatcaaccgacagctggaggcattggagaatgacaacttccaggatgacccccatgccggcctcccccagctgggcaagaggctacctcagtttgatgatgatgcagacacaggtgagatgggggttagggaggaggagcgagacagtgccgtgaagctccttgtcacccgtaactgcagatgatgccaatgagcgcagatgggaattgcatctttattgtgtttttaatttcctcattttgtgtgcatgcatatgtgtgtctgttcattcctgcacacgcttgcctgtggagatcagaaattaacttacaggagttcgttttctccttccagagttgaaattctggttactcaggttgctgagttcagcagcaggttcccttaagcaccaagccatcttagcagcccctgtttctttaaaactgcaacttattcagagagcaggcaatctgagaaatttattgatattaattaatatcaacttattgatattctaaagatccatcttctagcctgggggagccattgctcacatgtgcacgtgtgtttacaaatacacacactcacacacacacacatacacacatgacacaatgacacatacctttaagcccagcactagggacacagaggcaggtggatttctgtgggtcagcctggtcttcaattggagttccagaacagctagagttacacagaaaaaccctttctcaaacaaacaaacaaacaaaccatgttcagtcagcaggttatagacagagacaaaaggctgtcaatcatctggattggttgccatttccccttccctctgtgtcaggtgtcagccacatctctgagactagcgatggccgtgacctttgttatcatcctgtgtacttgttccttactctgctcatgtctgggctgggctccggcacttcttgaaccactgagtcaaattagtgtctgctggtgtcatctttgtactccaaggccatctgacagggagggggtggttagctcagcaaaccaaccaggaacagctgtatacgctaagctagaaacaccatgtatgtgcctgttgtctggtctgtttatagtagggagggttttgtttcttgagagagggtctctctatgtagaccaggctgttctggaactcacagagatccgcctgcctctacctcccaggtgctgggatcagcggtgtgcgccacgcccggcaagatgtttttaaactttaaataatctctcttgtttcatgtgtggcatggaggtcagaggacaatgtttgggagtagcttttctccttgtactgagggccagggactgaactcaggatggcagccttggcagccagcgccttttcccgccatcttgccggctcaagtgtgtcatttatacaaatgtaggcagagagggacagaccacataaaatatccctgcccttgggctggaggaaagggaaggcgtcgggactgtgggagggaaggctggcagtagggcggtgacttgggaattacttctggctttgccagtgcctggctcctgtgttgggagcatctgcctttcctgtctgtgtctctgtgcctcagtttcttcatatgtgtgggataaaaaagatggagcaatcaggtatagcgacccagcacacagaaagtttgaagccagagaactgccaagaatttaaggtcagcctggtctatatagtgagtttgaagtgggcttggggtaggtagtgagaccttgtatcaaagcaacaacaacaagactgtgtaatctgagcacctgagaggtgggaacagggagatcgatcaaagatcaccgtttatctacacagtgagttcatggccaacctgggcttcatcagctcctgtccccaaaataaaaagatggagtaggttggacttttaagtagctgacggtcgaccaggattgcagatatcagtcagcgttagtgcagatgtaggcagggagggacagaccccgattgtggtgtggataggcctctccaacaggactgtcacagccactgtccagcaactcagtgtccttcatcccccaggaaagaaaaagaagaaaactcgaggtgaccactttaaacttcgcttccggaaaaacttccaggctttgctggaggagcaggtgagagaaggctcagtctgaggttgggaccctagagagcacggggggggggggggggtgggggggggggtgggggggggggggtggtgagcaaggtcctgctcttcagcgccccttgtggacatcagctgcatggcggactggagaattccggatgcctgacccttcatgtccttgcagaacctgagtgcatctgagggccccaactacttgacagcctgtgccggccccccatctcggccccagcgtcccttctgtgctgtgtgtggcttcccatccccgtatacctgtgtgagctgtggtgcccggtactgcaccgtgcgctgcctgggtacccatcaggagaccaggtgagcccgagcctgacccagccatgcctacagtcttattccatgtagcatcccaggacctccctccttctccaatgggcttctctctctctttctgcaggtgtctgaagtggaccgtgtaaacctggcatcagggagagaaagtacccccacccttcccaacccaccccagctcattcacaaaggacagagagagggagaaactacccattcacccagcaaagctgtgccctgccgaccaaggaaggccacagggacccaggactgttgtgggggtggtccatccacagagtgctgtgataataaacggcctggtgaaaggaagcctgcctgctcattctggggtatttggaggtgaaggtcagggttgactgtcaggccagcatagactctgcctttcttcttgtagcctccattgccggccctctgtgtcatGGGAGGGTTGTGTCCCATTCTCCAGCTACTCTCAAGTCATCTCTGCCTTTCTTAGCTGCTGCTTTACTTTGGTTTTTGAAACAAAATTTTGTATAGCCTAGGCTAGCCTTGAATTTACTATATGTATAGTCATTAGGCTGAACTCCTAATCTTTCTGGCTCTGCCTCTGCCTCTGCCTCTTAAGCGATGTTTTTTAAATAGTTTTTCTTTCCATGTATCTG
This mutation is a 2990 bp deletion beginning at Chromosome 5 position 136,982,083 bp and ending after 136,985,072 bp (GRCm38/mm10).
JAX Protocol
Protocol Primers
| Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
|---|---|---|---|---|---|---|
| 48482 | TCT TCT TGT AGC CTC CAT TG | Wild type Forward | A | |||
| 48483 | CAG CAG CTA AGA AAG GCA GA | Common | A | |||
| 48484 | AGG AGA CTG GGG CTC TAG G | Mutant Forward | A | |||
| 48485 | Fluorophore-1 | CCG GCC CTC TGT GTC A | Quencher-1 | WT Probe | ||
| 48486 | Fluorophore-2 | CTC CTG TCT TGA CCC CGG | Quencher-2 | MUT Probe |
Reaction A
| Component | Final Concentration |
|---|---|
| Kapa Probe Fast QPCR | 1.00 X |
| ddH2O | |
| 48482 | 0.40 uM |
| 48483 | 0.40 uM |
| 48484 | 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.