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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
>chr10:79966827+79966919 93bp TACCCAGGTCACTGGCATC TGAGCCCGTTAGGCTGACA
Mut= 92 bp
Wt= 93 bp
Fam=Mut
Hex=Wt
Sequence
Wt Sequence (deletion in lower case):
TGCAGCTTCTCACAGCCCAGTGCCCCGCCCCTTGATGGCATGGGGTGCACACAGTCAAACTTTCTGTATCCTGCCTCTACAAGTGGCCCTGACCCTGTGGCAAACTGGACATGGGACACCATTTTAACCCACATGGCCTTAGCCTCCcaagtagtggaaggacaccgcccccattgcccctctgtgtgctgagctggaggctcacacattttgagccagcaggggcctcttctggggacagggtagaatcccattattgtcagacactgaggacacagccagagctggccaagtgcacagtgatgagggcctgcagccttcctggttccatatgccaatggccctggagcaggtggacggggttcttatgggttggcatattctgtgttgcccctgaccttcagtgcccggcctctcacaggtctgcaccgggaacctcctggtcatcctcagccggcactaccaggacgtgtcctgcctgaagtttacaggggatggcagtcattttgtctcagcgggcaaggactgcttggcgctggcctggagcctctgcaggtagggcatgtgccagagatctacccagaaaggctatggaggtctcagggcaggcaataggctggtgactactgaggtatcccattcttgggggcttggggaggcaggccaggggctttccacgtggcctcactgctgttgtgtgcccagtgtgctacaggcagacccatccaggatccttgccccaagacatgtctggtcccagcacacgctcccaatcacagacctgcactgcggcttcggaggccccatggcccgggtggccaccgcttcattggaccagactgtgaaggtagcatccccagcctcgacccatgaattcctggcagccactggatctcagctaggagggagggagactggcatctcccttgccttggctgtttgggctccagacagcccataatccctatgcccgaccaaggacacccagagatggactgtccagcctgtctgctggaaattcccactcgtccacgcctcagtggccagatgcccttgagtgtgtagggttgtttgtttgttattggcacagtgttagagatggaacccaggccctgcatctgctagaccagtgctctacctgagcctcacctcagccctcttttatcttctagttttaaacagttttttgcttgttttttgagactgtctcattgtgtggccacagcttgcctggaacttggtttgtacaccagattggctagggattcacagacccccttgcctctctctgcctaggagcacagggattgagggcaggattggagacatattccctggctggggtgcctgtgattctgtgggggttcagaggctggtagcatgggcatgatggtgcgatggcccccatgcctcctggccccccgacttggctgtgtgcctggatgtccacagctccaggcttcccattcctgcagctctgggccatctcctcgggggacctgctgctgtctgtcttgtttgacatgggcatcacatctgtgaccatggacctggccgagcaccacatattctgtggaggcagcgatggctccatcttccaggtggacctgtgcagctgggtgagtggccctctgggactgagggagcccacagtcctttgccctacatcccaggcacttaacgacctgtctctacccccagcccggactgagggagcacagcttccagcctgagcagaacacagggaaggtcttcaaaggacacaggtagtcacctcaggaggtggggttagcacagtgcccacaccgtgaagcacctaacccccatcctcctctcccaggaaccaagtgacatgcctgtcagtgtcaacagacggcagtgttctgctctctggctcccacgatgaatcagtgaggctgtgggacgtgaagagcaagcagtgcttgcgcacagtcaccctcaaaggtgtgcccgaggcagcgtggccacacagggacaaggcagggccctgtcctgccagccaggcccctgatgttgtgttattgtgtccttcaggcccagtgaccaatgcagccatcattctggccccacccagcatgctgaaccctgaattcaggcccagcctgcccctgccccacttcaacaagcacctgctgggggctgagcatggagatgaagcccagggtgggggcctgcgcctgcagctagggctccacctgcaggtaggacccacagccactcttctagaacattctatactccagagttccttctgctgctgctggtgtgcctgtctggcatcaacagcttctgaacccttgctacccaggtcactggcatccactgctgctcttgccatccgtccccacaacatacgcctgtCTGAAGGCATACTGTGTCAGCCTAACGGGCTCAGCTGACTTGTCACATTGCTCTCAGTGAGTGGCCAGAGAGAGAGTCAGAGCAGCCTGAGGTCAGGAGACACCTCACCAGGAAGGGGCTTCTACCCAGTGTCCTCAGTGCTGCCCCTGGGGCTTTGACCTAGGTCTGTGCCACTCTACAGTGGCAGGCCCTTT
This mutation is a 2235 bp deletion beginning at Chromosome 10 position 79,964,652 bp and ending after 79,966,886 bp (GRCm38/mm10).
JAX Protocol
Protocol Primers
| Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
|---|---|---|---|---|---|---|
| 44530 | TAC CCA GGT CAC TGG CAT C | Wild type Forward | A | |||
| 44531 | TGA GCC CGT TAG GCT GAC A | Common | A | |||
| 44532 | CTG ACC CTG TGG CAA ACT G | Mutant Forward | A | |||
| 44533 | Fluorophore-1 | CAT CCG TCC CCA CAA CAT A | Quencher-1 | WT Probe | ||
| 44534 | Fluorophore-2 | ACA TGG CCT TAG CCT CCC | Quencher-2 | MUT Probe |
Reaction A
| Component | Final Concentration |
|---|---|
| Kapa Probe Fast QPCR | 1.00 X |
| ddH2O | |
| 44530 | 0.40 uM |
| 44531 | 0.40 uM |
| 44532 | 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.