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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
Mutant= 85 bp
Wild Type = 88 bp
>chr14:55575465+55575552 88bp ACACTTCCACCCACCCAAG AGCTGGCAGGCAAGAAAG
The WT Probe (63981) anneals over the nucleotide sequence containing mouse genomic variation rs236536210 and the Mut Probe primer (63982) anneals over the nucleotide sequence containing mouse genomic variation rs214770140.
Sequence
Wt Sequence (deletions in lower case):
AGCTTTTGCTTTTTGCCTGCTTGCTTTCCCCCTCCCTGGCAAGTTCACGTGTCCCGTTGCTGAGGCATTCCTTATTGCTCTCAGATCCTAGGTCTTGGGGACTCTAGCACAGTGACCAACGGAGACATCTAGCCTCATGGACTGAACAGCCACCTGGTCGATATTCTGTAGGAAGATAGTTATTTTTGGAATTAGGCAGCCCACCGCCCATGATAAAGCCCTCTAATAAATCCTCTTTTGATTTAGATTGATTCTATCTGTTCTGCTCCTCTAGAAAACTCTGATTAATACAGACATCCAAGGTTTAGGTTGGTTTGATGACCTAGATTTCCCTGTGCAGGAAAAAACATGGTAGAGTCTAGATGAGCCTAGATGGGCTTCAGCTGGCCAAAATAAGGCAAGCGGACACAAGCTGGCCTTGGACAAGGACAGCTCGGAACGGCAGGCCTTGCCCTGAAACACCAACTAGAATGTGGCTGGAACTGAGAGCACAGAGCTAACAGGAAAGGACACAGGCTATGTCAGGACACACTTCCACCCACCCAAGGCCAGGACCATGTTgaccgtcagtcctcaaactggccccttgctggcccccctttcttgcctgccagctgccaacagcgctctgccctgtgtctaccacacctgtcactgcctatccttgtctgagggaagggggggggggcattagcccctcctcagcctccagcagagcagacagttagtggggaggggagggaacatggaacgggggccgacggtgggggcagggctgggggccgggacccgagtccgagcactgctgggatgcctggtcaaggtgctgctctgggtggcctctgccttactgtactttggaagtgaacaagccgcccgcctcctgggcagcccttgcttacggcgcctctaccatgcttggttggcagcagtggtcatctttgggccccttctgcagtttcacgttaactctcggacgatcttcgctagccacggcaacttcttcaacatgtgagtccactgaagtccataggagctgcctctctacaccctgggatcccagcttccttcttcaggcctctgtgttctgttgccagtctgaatgctaaaaataggctaggaggttgagaaggtcggggggaggagagggggaggggagagggagagaaaagagcctgggggtgccggggaagtaaaggacggaagaggaaaggtggaccctagttgccctgtggtcctgcaaggagaagggcaacattgaaatgataggggttataaaaaaaaaaaaaaacctaatggataatgtatgggacacaggtcaggatggggagatcggacactggggatctcaactagcttttccacttcctgtccacagaaagtttgtgaattcagcatggggctggacatgcaccttcctggggggctttgtgctgctggtggtgttcctggctacacgacgtgtagcagtgactgccaggcacctgagccgactggtggtgggggcagctgtatggcggggggccggacgtgccttcctactcatcgaggacctgacgggttcctgcttcgagcctctgccccagggcctattgctgcatgagctgcccgaccgcaagagttgcctggcagccggccaccagtggaggggctacactgtctcctcccacaccttcctcctcaccttctgctgcctcctcatggctgaggaagccgctgtgtttgccaagtacctggcccatgggctaccagctggcgccccactgcgtcttgttttcctactcaatgtgctgctgctgggcctctggaacttcctgctgctctgcacggtcatctatttccatcaatatacccacaaggtggtgggtgcggcagtaggcacgtttgcctggtaccttacctatggcagctggtaccatcaaccctggtcccctgggatcccaggccacgggctcttccctcgatcccgctcaatgcgcaaacataactgaaagaaataaaagcataccaggcctggctctgtctcctccgattcttttcgggacgagagagagaatgggaagggtaagatgctcaagttccttcctcatcatttcttgaagttgctgacctgtgttttcctgtacgtgacttccatccTGTTGGTTTCCGGGTCTTTCGGGGGCTGTGATGTCGAGGGACGGTACCACTAAAGCAGCCTTTAGATGCACCAGCTTCCCTCTGCGGATGCCAGGGCTACACAGAAAAACCTGGTCTCGGGAAAGAAAAAGTTGGAAAAATATTTCTGGATGTGTGAAAATTACATGAAACTCAAAATCTCCAATCCATAAGTTAATAATTCATTTATTTATTTTATTTTATTTTTGGTTTTTCCAGACAGGGTTTCTCTGTGTAGCCCTGGCTGTCCTGGAACTCACTCTGTAGACCAGGCTGGCCTCGAACTCAGAAATCTGCCTGCCTTTGCCTCCCAAGTGCTGGGACTAAAGGCGTGTGCCACTACACCGGGCAGTTCTATTGAAAGTCATCTGCACTAATTAATTTCGTGAGCTGCTTTCCTTTC
This mutation is a 1578 bp deletion beginning at Chromosome 14 position 55,575,498 bp and ending after 55,577,075 bp (GRCm38/mm10).
JAX Protocol
Protocol Primers
| Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
|---|---|---|---|---|---|---|
| 63978 | ACA CTT CCA CCC ACC CAA G | Common | A | |||
| 63979 | AGC TGG CAG GCA AGA AAG | Wild type Reverse | A | |||
| 63980 | TAG TGG TAC CGT CCC TCG AC | Mutant Reverse | A | |||
| 63981 | Fluorophore-1 | ACC GTC AGT CCT CAA ACT GGC | Quencher-1 | WT Probe | ||
| 63982 | Fluorophore-2 | CAG GAC CAT GTT TGT TGG TTT CC | Quencher-2 | MUT Probe |
Reaction A
| Component | Final Concentration |
|---|---|
| Kapa Probe Fast QPCR | 1.00 X |
| ddH2O | |
| 63978 | 0.40 uM |
| 63979 | 0.40 uM |
| 63980 | 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.