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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= 115 bp
Wild Type = 116 bp
Fam=Mut
Hex=Wt
>chr12:30890262+30890377 116bp CCTGCTGGCTATCTGTAGTGG GGAAGTCGACAGCAATAGGTT
Sequence
Wt Sequence (deletions in lower case):
CTGGAACATAACCAAAAAGAGAAAATTGTTTTTGTCATCCTGCAACTTTATGGCCTCCGAGAGAGAATAGACTGGTAATATTTGGAGCTTTAAGAGTAGTTAACGCTGCTTGAGAAGAAAAATCATGTCCCGACCTAAGTGTCCACACTGTATTTTGTGTAACTACCAAGTGTTCTCCTGTTTTATCcagtgcattgtagtatggataatgcaattctcagaatttcctccacactcactttatgaccacacttcataggttctctgttcaagtaatacgatttctcacccctcgtagagattgttcctcgagatctaaggatgaaggacaagtttctgaaacatctcacaggtgtgtgtccccttcagtcctctgccctccctgctcctttgtgccctgagtctttgtgactttcttcctctgtgttccaggtcctctttatttcagtccgaagtgcagcaagcactttcacagactttaccacaacacgagagactgcaccatccccgcatgtaagtgagcacccagtagacaaaactccactctttcctcaacgcagtgggtctgctctgtgtcaggcgtgggggcctgagtgtctccacctgactgggttgggacaggatgcccagacattggagcagatggcttaaactgtccagcaaccagtgccactgatgataacccattggatgtaatcagctcttgcagtgggttgaccccttccattcacatcctcatttgtttatgaactctggcacaaagtccatattctattcagctgaacatcacacaagtcaggtgtgagttgcttgggtgtctgctggtggtcagagtgttgaagtaaccagcctggttggcctgaggttctttgatcaaactattctgaccatctccatggctctgactagtgtggttttcatcccatcaataataaagccctcagcacccaaggattttatttgctattccaaagcttttcttttggctgtaattcttcagcccagtgacatggggctcatacattggccccgtttctcatatgtctttctttcctttaaaatttcttttcattttcaacaagcttcatctatttatctactgtaatatagttcactcccccgaaatggttccttacactcttcataaagccattcttttaaatgggatgatgatctttgcttgatttaaaactgaaagaagaaaagccatgcagacacaccaggatctcgctggcagaggtgacagaggaccgaacccagtcagcagactgaagtggcctttttgcttgccacaaatataccggttacatgaagtagtaattttcttttttctgtttaggcagcagagcttttctgaataatgatgtctgtcatttacagactgcttcagtgtgacagacactgctaggcaggctcatgccctcacatctgtgggtcttggcccagcagggctgcttcaggcgcccttatcagacagagtcaggcctgaagcccaaggtgagcagacacgctccctgaagaattcgattttccatttgaacttgccataagttccccttctctcattacctcccctgctctcttgtgcactgaaaccatcttctgcttgagaattaacttctttcttgaggccatagaaccttttttattgtttaagtgtgtacctatctattttacaataaatattctcatacaatgtaattctagattctaaattttaacatgaccttctagcattaaaacctatccaattagagattaggagacaggatttaaatggcttgtcacagaggcatcaacaagaccaggctcaaggctcatgctccactctacagtctcttcaagcttctcccagagagctgaactatgctccacactgtcgtatccttttccaataatatacagagacttctggccacatctcaatctgagtccccagaggtggaaggttgacccatttgtgcattgagattagcccctcccgacttcttcttccacaaagaacagagcagtaggtagctcttccagggtgctttaagatgttcagaaattcactgaaatcctggggcgctagaatcaatatggaccagagagcccaatcttcttagatagccatagtagcttggcggaatgaaaacaaagtctttcttctttccgataaacagaatatgttctacctacattcgtattagtggttgtggggcattgtttgagacgtggactgagataggaacagctaggatctccacaggatacaaatgcaagtatttacagtttgttaattaagtcactcctggactcacaatctactccatcccatttaggaaaactgtgttccaaacagtgagcaagaaaagcaagaaagtcaaaatctaggagatgaagtccaaacaagttaagctctctttcctctttgaaccaaaagtatatatgccatgaaagtttcgaaggtaacttgagagtgtgtagccattgtcattgtcactcattcagattctcctttgagggtgaatacttcctgagaagtagactgtagtaaatcataagtcacaaaggcttttcacataagtgtgtcctggggaaattcttctatagatgtggtaactgagccttcctacatatttcttgatggagcagaagaaggaagtgtggaatcataggaagccacacagttacacttcttgttacacttgaaattttgctttttaatcccccctttactgtttactgttgatgtttactgctacatctattcccatttctcttttttactttttggaacatactcttgttaagttgctcagggtggcctcaaattcattcagtagcccagaacttatcctcctgccctgcttcttgcatagcctcccacctcgatccctttgacccactttctttaaaacatcactgtaaaagtctgtataaggctcatctcaggtggggacttggtctgcatagaatctgcattcaggaggaatgtaaaaagcctatctcacttgaagcaacgctcgtgtcatttctgtgaagctggcagccatagttcagaggactgtgtctgtcccctctgtaaccttcatcttatttctgcttgtaatgccaagaaagtaagcaacttagaactcatgattccagataagactctattgcttcagaatattttttttaaaagttagtgttttatatacttgcaaaacaaacaaatgaacaacaacaaaaagcagcctatgtttcattttagtgttgtgcagacaactgacgagggtccatggtttgttttcactgcagactataaaagatgtgccaggttacttactcggctggcggtcagtcccatgtgcatggagagataaggtaagctcccaaagaatcgtggtggccacacagcaggtgtcaggatcccatttaggtcataaaacacacatggaattcagtcatgcattttctacagtagcctcactctgagcaaaagacagccaactcacctcaaaacattggccatgagtagaaggctccccctgctggctatctgtagtggacattgttactggaagttgatattagcacttagctgatctcagCAGAGGCAGTTTACATGAGGACACAGGTTTCAACCTATTGCTGTCGACTTCCAGGTTCAACACTTACGTTACTGACACTTATGGACACAAATGTCCTTCGAATTTTATTTAGTAAACAACAGAGAGAATGTGGTAGGCTACGAACTGTGGCTAG
This mutation is a 3391 bp deletion beginning at Chromosome 12 position 30,886,935 bp and ending after 30,890,325 bp (GRCm38/mm10).
JAX Protocol
Protocol Primers
| Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
|---|---|---|---|---|---|---|
| 62623 | CCT GCT GGC TAT CTG TAG TGG | Wild type Forward | A | |||
| 62624 | GGA AGT CGA CAG CAA TAG GTT | Common | A | |||
| 62625 | ATG TCC CGA CCT AAG TGT CC | Mutant Forward | A | |||
| 62626 | Fluorophore-1 | TGA TAT TAG CAC TTA GCT GAT CTC AGC | Quencher-1 | WT Probe | ||
| 62627 | Fluorophore-2 | CTC CTG TTT TAT CCA GAG GCA GTT TA | Quencher-2 | MUT Probe |
Reaction A
| Component | Final Concentration |
|---|---|
| Kapa Probe Fast QPCR | 1.00 X |
| ddH2O | |
| 62623 | 0.40 uM |
| 62624 | 0.40 uM |
| 62625 | 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.