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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
>chr7:102681802-102681958 157bp CTGTGGGAGTTAAGATGACTCT CACTGGAGCACTCTAGGCTGA
Mut= 168 bp
Wt= 157 bp
Fam=Mut
Hex=Wt
Sequence
Wt Sequence (deletion in lower case):
TGTGTTGGGTAAGACTCAAGCTTCTGCCATGCTGATATTCCTGGCTTTGGACCACACTTTCAACAGCAAGTTCTGGGAAATAATAGAGAACAGGACTAATGAGAGGAGAAATAGTCCTGGCTAGGAAAGTCAGGTTATATATCTTTATAGACGCTTGAGGACTGTGGGAGTTAAGATGACTCTCAACCCAGAAAGCctccctgcatgtcacatgggcagaggctgtgccttccgtggcagtccttttcctcttatgttatcccagccagctgcctgaggaacattgggaagcccctgtcagcctagagtgctccagtggcctcctgatggggcacgctcatgatccttctgtctcttgcagtggaaactccagcaggctctggagcatctgaggaaagaacaggaagaagcctggaaactggaagtcagtgagaaggaacaggctgccatctggaaggcaggttgcacttgagaagggggctcactcacatcacccctctcttctagagccgacggtggagtcttcctcacgtctgtgaggcagagtgagggccttcagcctcccttttcctaaggccacataacttctcaggtccagcttatagctttggggtagcagtagcctctgatttcatatcttcacgtatgaggaaggaatggcgccttgcacacgtgagctcctaggagtatagcttatagagtggtgatctaacagtttctgtctcactttcctgagtgacgacggcacagctgctaagactttggttgcctaaatattaggaacccccctcagaaatatatttgaaaccaataggcataaaactctctgaacttttggtccatgtgatctaaaacaatacaataaacataaacacacacaaacacacacaaaacaaagaagaaaaactatccagcagtcttgttagcaaccatcagtgtaaccatctgactggttttctgaaaatgagagctagtccccatcatgccaagataatatcatgtaccaggatggtggtacatgcctagaagtcagccttgggaggcaagggcagagaactgctacagtttcaagtccaacctggtctacatagtgaataacagtccagggtggcaggggctagacactggagccctgtctcaagaagaaagaaagaagataacctagattctgttgcagttgataaaagtagtgcagacagattagaggtcactcatccagacatctgaactgaaagtgctcccaaatccaaagtgtcttatagccaacatgctaccaaaagtagaaggaagtttcgctgaatgataggttatactcagaatgcaagcacactaaaatcattttacaaaattactttcaggttctctgtacaagatacatgtgaagcatggatgaatttcatgttcagagttggatctcattgccaaagtatctctttatattatgtaatctaaaatccagaaaagttccaagtctgaaatactctggtcccactcattatttgtaagaaaatacactacttatgaaaggaccaaaaataatgcctggggccatggggagttaggaacagaaaagttagaccctttgtctttgccttcataacacccagaacatggttgtagcaaagcatatccatcgtgtgtgtgtttgcacatgtcagtgtgctccatcccacgtgacccttaccctaaaaagaccagtgctaattccagtcttgactccagacacagatggaaagacgaaagcagagcatccgatgggagtttgaaaaatatcggcagttattaaaggaaaaggagctgccctgccagcaggcagaagaggaggcagctgcagctcaggctagcctcgagcaggagaagggagagacagcaagcaaactggagttgaggcgcgaggcgatcatccggcagagccaggtcctgtggagcatgattgtggagctggaagagagatcccagagacctgtgcgctggatgctgcaggtgagcaacagccttccgtgggatgcctgagcgagaactggagacggaggctccaggggaaagtattagctagttcttcacggaccctatcatcattctgtccaccagctgaggaccctgtctgccttcagctgacacatgctgctctcatcctgattgggtaaaagacctgacccttaattttctcataggccagaagtaggagtacaccgggtacttactctatagcttggcagcattctagtcccttccatcctggtcatcttgagatggagtctgaaggtgttccatggagccaggtctgctctgctgtcctcccatgagcttctccccacctctaaagtctgtgtaaccctgtgagaaagcccctcccagagtttacgcctgtctagtgtctctgacgcaccgcacattcatgggctgcattttttttctcctagggtattcaggaagccttgaacaggtacgttcccatccctagagactctgtgaaaccctacaggagatagagaggcctggccagagccagggatggagaaggaatgtagactgagtacctacagaaggcagatgctcaccgtCTTACATTAGTGAAAGAAGATTTGTCTCCTTTTTTGCACTTAATTGAATTCATTTCAGATTTTTCCCAATAAGAATTTTAAGTAAAGCTGAAGTATGTTACTTCATGTAAATGGTCCAAAAATTTGATGCGTGAAACTTCTAGCTCAGAAGATCACCCTTTCTCCTTTGTGTCACATCTCTTTCTCTCC
This mutation is a 2368 bp deletion beginning at Chromosome 7 position 102,679,556 bp and ending after 102,681,923 bp (GRCm38/mm10).
JAX Protocol
Protocol Primers
| Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
|---|---|---|---|---|---|---|
| 46838 | CTG TGG GAG TTA AGA TGA CTC T | Common | A | |||
| 46839 | CAC TGG AGC ACT CTA GGC TGA | Wild type Reverse | A | |||
| 46840 | CAC GCA TCA AAT TTT TGG AC | Mutant Reverse | A | |||
| 46841 | Fluorophore-1 | CTG CCT GAG GAA CAT TGG GA | Quencher-1 | WT Probe | ||
| 46842 | Fluorophore-2 | AAA GAA GAT TTG TCT CCT TTT TTG CAC T | Quencher-2 | MUT Probe |
Reaction A
| Component | Final Concentration |
|---|---|
| Kapa Probe Fast QPCR | 1.00 X |
| ddH2O | |
| 46838 | 0.40 uM |
| 46839 | 0.40 uM |
| 46840 | 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.