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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
>chr9:121826844-121826926 83bp CCTGGGAATTGAGAGATGTT AAGGCTGAGCACATTTATCT
Mut= 101 bp
Wt= 83 bp
Fam=Mut
Hex=Wt
Sequence
Wt Sequence (deletions in lower case):
ACCAGCCAAAGCTGGTATCGGGCAGATACAGAAAGGTCAGGGGCTATGCCCTGCTCACTGAGGTTCATAGCTCACTCTCTGTGCAGTTAAGATCCTGGTATCTGATTATTTTTCCAGGTCTTAGTGATGTAGCCCAGGCTGAGCTCCCTGTGTAGCTGAGAATGGCTTTGGACTTCCAATCCCCCTGCCTCTGGCTCCTGACTTGGGATTACAGGCATTGCGCCCAGCTGCCTTCCGATCTTATAGTTTGCCTGCAACCTGCTGatgctctgaacagctgtgcagatttgaggtgcaggttgtgtttttaaatggaccttgcgatttctttcattttaggggcgtctgggatggctggagatctagttgccaccaaaattgttgaactgtccaaaaagaaccgggggctgatggccgagtcagagagcgcaaaggtcaggataaagcagctgaccaatcgcatccaggagctggaacaccaagtaagggcctgggctgctcttcctggggagtgaactccagagagaggccacacttggaggtcttgggtccttatggaggtcacagggcacagctgtttggagtgagatgcctggtgtctgccacatgcaaagagaggaagtatgtcaggcagctaggggacactggccattgagaagaagcctggcatggtctggctaagggcgatgaacacggagtttcactgtgagatctgcatctgttcgcatgccccacacctgtcttggttccccatccaagtggacagcgttggccctgctgccaggctgcctgggaattgagagatgttgtgtttaactgtgtaggacagagcctagcatgtagaaagccctagataaatgtgctcagccttctcccagaaacccccgttcaactgaagccacatccagaagcagtgacatgatgagttctgtctcccccatttcgctgtcccttcctcccacctagccacagacctgtctgctccagcagggcacagccccatggttctggttcctgaagagttggaatggctcctcagactggagcctctgtagccttttctgagtgcaaagtctattttcgagtctctgcatgatccaccctggtcctcagagctggcactaaggaagagtttattcattctgcgatgtgtttctggagagttggctcatgttctgagccactcactggagggaatagagtaggcatgggtggttccactcctgggagatggtgctgtgtgggagtgtggcacagcacagccagggcaaaggaaaaggagcgacaccttgacaggcaacagtggcaggagctgcttgaaggggctttcccagcaggcccagccagaggctgtgggaacaacatccactcaaagggccttgagacccatagtgccaaatgggcagaaagtacaagagaccagtatggccaaagcctatataggatgatgtcagggatgtgagaggggaggccatgccccaaatcagattgaccaccatgtatgaagaacaattgaaaagtcactaaagggtagctgcctacagcaggttctgtcacaaggctgtgcccatgtggtagccaccctctgtctcacctgaaacccaaagtctttggttcctttattctctgccttctgggctgagagtgtcttaatttgagttttattgctgtgaagagatgtcctgaccatggcaactcctataaagggaaacgttaactggggctgacttatagttctgaggcttagtctgttatcattgtggtggtttgaacaggaatggtatccaaagagttgtgtgtgaatgcctggccatagagagtggctctattaagtgtggccttgttggaggaggtgtgtgtgtcactgtggaggcgggctttgagatcacagatgctcaagctatgtccagtgtggtatggtctcctgctgctgcctgcgtatcaagccgtagaactctcaggtccttctccagcaccatgtctgcctgcatgtcactatacttcttgccaagacaataatggaatgccagtctaggggcctgtgtgtgccaacaatgtacaggcacatttaccaagtgaatcaatagccagccccagtgaaatgttttcctttgtaagagttgccatggtcatggggtctcttcacagcgatagaatcctaactaagacaattgtcatagcaggaagcatggcggcacgcaggcaggcatggtgctgtagaggaagttctgagttctacaactggattcacagacaacaggaagagagagagtgacatcaggcctggcttgagcttcggaaacctcagagctcacccccagtgacacacaagagtgcacctcccaatggtgccatttcctaagagtctatggggccattttcattcaaactaccctagaaagtatgactgaactgtggtcatttattaaggtcttaacagatctttgggctgcccttcctaggaaatagggttgattaccaggcaaacctctcctgtcacaagtagcttggaaaggagcattccttagtgttccacatgtgagcaagaactcagacactaggacaggtggcccacacatccagcagcactgggtgcttcggctggctgtgcagctgctgagcagaaccaagtcttagtgccgcgtttgcttttccagctgcagatggcgtcagccaagccaccatccaagggggccactgatgcaggggctaagccactgaagacccagacgggagacagagctttggtatgtgttctcctttatgctgcgtctgtgaggtagccatgacctgggcctccccttccccagtgtgctctcagatactctgccacagttcccagccaagggctcttctctgccattGTAGCCTTAGAGCAGTCCCAAGCAACAGATCCGGTCTATGTCTTTTGCCCAGAATGCCTGGCAGGGGCCAAGGAAGGCCCAGATCTAAGCCACTTTGCATGGGGGTCAGGGGACTATACAGCTATAGCCTCTAAGCCCTGGGAACACAGACCAGGAGGAAAGGATGGTCCACTTAGATGCCTGCTCCCAGGCTGATCCGCTCTGCAGTAGTCTCTTTACAGGGTGACAGCTACTACTCCTTGCTTTGCAGACAGACAGGCTTGCTCCTGGTTGCCCAGAACAATTCTGTTCTTTTCCCTTCAGTGTGTGTGT
This mutation is a 2,600 bp deletion of region Chr9:121,824,851-121,827,450 (GRCm38/mm10).
JAX Protocol
Protocol Primers
| Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
|---|---|---|---|---|---|---|
| 71593 | CTC CTG ACT TGG GAT TAC AG | Mutant Forward | A | |||
| 71594 | GAT CTG TTG CTT GGG ACT G | Mutant Reverse | A | |||
| 71595 | Fluorophore-1 | CCA GCT GCC TTC CGA TCT TA | Quencher-1 | MUT Probe | ||
| 73380 | CCT GGG AAT TGA GAG ATG TT | Wild type Forward | A | |||
| 73381 | AAG GCT GAG CAC ATT TAT CT | Wild type Reverse | A | |||
| 73382 | Fluorophore-2 | ACA GAG CCT AGC ATG TAG AAA GC | Quencher-2 | WT Probe |
Reaction A
| Component | Final Concentration |
|---|---|
| Kapa Probe Fast QPCR | 1.00 X |
| ddH2O | |
| 71593 | 0.40 uM |
| 71594 | 0.40 uM |
| 73380 | 0.40 uM |
| 73381 | 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.