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Notes
Taqman Probe/Endpoint protocols use a different analysis than qPCR. Please see your equipment manual for more information about Taqman endpoint analysis setup.
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
>chr16:97949845-97949940 96bp TGCCATAGGACATTCAAGAC CCTGCTACTTTGTGATAGGT
Mut= 92 bp
Wt= 96 bp
Fam=Mut
Hex=Wt
Sequence
Wt Sequence (deletion in lower case):
ATGAATAAAGTAAGTGTAACTAAGCATCAGATGGGTAAGGACCCTAAATGCTCAACTTAATCTTCAAGTGTCATCTCTGTCTATAACTATTAATTTTGAGCAGGCCTTTTGGTAGAATACAAATGGTGATGGCCTCACCGTCTGTCTTTCAGATAAAGTGCAGGATggagggactgctgcattacatcaacccagcacatgctatctctctgctcagtgccctcaatgaggagcgcctcaagggacagctgtgtgacgtgctcctgattgttggggaccagaaattccgagctcataagaacgtcttggctgccagcagtgagtacttccagagtttattcacgaataaggagaacgaggcacagactgtctttcagctggacttctgtgagcctgacgcttttgacaacgttctgaactacatttactcttcttccctttttgtggagaagggcagccttgctgctgtgcaggagctggggtatagccttggtatctccttcctgaccaacatcgttgccaaagcccctcaggctccttttccagcctgtcccaacaggaaaagagtgccagtggaagatgatgaaaccagctctcaaaagcgaagtgtcattgtgtgtcagggcagaagtgaagtgccagggaaagccagtggtccagccatgcaggacctcagccatactgcccgggcctcccccagtgcttcagtcaagaccaccatcagtaagccacatgtggccaagccgccagagcagcttcatagtctgcccttaactgaaaagagctggacaaaggatagtgctgcagtatatgcaaagtctctggaacaggctggggctttggatgatcctaataggggcagtttggtaaagagaaatacagtcctgcccccaaagccttcacaggacagggaggccacagatgataaaccaggggtgagcagccagcttcccaaggggaaagctatagagctggctctgaagagaccacggccccctgttctgtctcttcatagctcatcagagactccatatctcctaaaagaaactagcaaaggaggcggtcagggagaggataggaacttgctctactactctaagctagggctggtggtcccttctggtgggcctgcttctgcaaaccagagcattgacagaagtggcccactagtgaaaagcctcctcaggcggtcactgtctatggacagccaggttcctgtttactccccctccatagatttgaagtcatcccagggatcatccacagcggcaaatgaggcaccgggtagtgtgttctgttcgatgtctcaaaagtcatctttaaaagattgcagtgaaaaaaaagctctggatgacaggcctcaagtgctccagcctcatcgcctcaggtcctttagtgcttcccagtcaacagacagggaggaagcctcccctgtgactgaggtacgcattaagacggaacccagcagcccactgtcagacccctcagacatcatccgggtcactgtgggaggagacgcagcagcagcagcagtagcagcagcagcagcagcagcagctacgagagacctgcccctcaaaacagaggaagaccagagagatatgagcagactcccagcaaagagaaggttccagacagacagaaggtcacccttgaagaaggcaaaggcaaatgaacacgggcctgctgtctcagaagagaactgtgaagagggcaggagccctccttcccttgacagcaacttcccagattctgacttaaacagagaggagtttggtgagttggaggggacgagaccaaacaaaaaatttaaatgcaaacattgccttaagatttttagatcaactgcgggtcttcaccgccatgttaacatgtaccataacccagagaagccttacgcttgtgacatctgtcacaagaggtttcacaccaacttcaaagtgtggacacactgtcagacccaacacggcatagtgaagaacccatcgccagcctctagttcccatgcagttttggatgagaaattccaaagaaagctgattgacatagtgagagagagggagattaagaaggccctgatcattaagctgaggcgcagcaagcctggcttccagggacagagtagctccccagcacagcaagtcatcaagaggaacttgcgctcccgagccaaaggggcctacatttgtgcctactgtggcaaggcataccgctttctctctcagtttaagcagcacataaagatgcacccaggagagaggcccctcggagtaagcagagctgctaagccaaaagagcgggctctggcacgcgcggtagagaacaaggaggtttacccgtgccgcctctgtaatgctaagctctcttctcttctagagcaaggcaaccacgagcgcttgtgccggaacgccaccgtttgcccttactgcagcctcaggttcttctctcccgcactgaagcaggaacatgaggacaggtgtgagtacaaaaagctgacttgcctggagtgcatgcgcaccttcaagtcctccttcagcatctggcggcaccaggtagaagtgcataaccagaacaacatggctttagcagagaacttcgccctggccaccctggaccacaacggtgaagtggcagccgcttccaggcctcagactgagcccagcaaggtaaaccatgtggccactccaaaagaggatacagcatttagcgactcttcagagcaagtgaactttgattctgaggattcctcctgcctccctgaagacttgagtctttcaaagcaactgaaagtccaagtcaaagaggagcctgtggaggaggcagaggaggaggctcctgaggctagtgcagctcccagggaggctggccccagcaaggagaccggcctatggccctgtgagaaatgcggaaagatgttcacggcacacaagcagctggagcggcaccaggagctgctgtgttctgtgaagcccttcatctgccacgtatgctacaaagccttccgtaccaacttccgactctggagtcacttccagacccacatgtctcaggctacagaggagcctgtgcagaaagaatcagaggtgtgtcctgtgcccacaaactccccctccccaccacctctgccacctccgccacccttgcccaagattcagcccctggagccagacagccccacaggcctgcctgagaacccaaccccagctacagagaagctgtttgcaccccaggagtcagataccctcttctaccacgccccacccctttcagcaatcacatttaaaagacagttcatgtgcaagctctgccataggacattcaagacggccttcagtctttggagtcatgagcagacacacaatTAAAGGACCTACCCCTTTCACCTATCACAAAGTAGCAGGCCCTTGGCTTGTAAACTGTGTCCTAAGACAGAATATTTGAAAAAATAATAATAATAAAGGTTGGAAATTATTATGTTTGAATTTTAAGTTTGGGATGAATTAATACTGGCTAGAAATGACCTCACAGCTTTCTGGACCTCAGCAGCAGGGGTTT
This mutation is a 3,196 bp. deletion of Chr16:97,949,884-97,953,079 (GRCm38/mm10).
JAX Protocol
Protocol Primers
| Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
|---|---|---|---|---|---|---|
| 74858 | TGC CAT AGG ACA TTC AAG AC | Wild type Forward | A | |||
| 74859 | CCT GCT ACT TTG TGA TAG GT | Common | A | |||
| 74860 | AGA ATA CAA ATG GTG ATG GC | Mutant Forward | A | |||
| 74861 | Fluorophore-1 | ATT GTG TGT CTG CTC ATG AC | Quencher-1 | WT Probe | ||
| 74862 | Fluorophore-2 | AGT GCA GGA TTA AAG GAC CTA CC | Quencher-2 | MUT Probe |
Reaction A
| Component | Final Concentration |
|---|---|
| Kapa Probe Fast QPCR | 1.00 X |
| ddH2O | |
| 74858 | 0.40 uM |
| 74859 | 0.40 uM |
| 74860 | 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.