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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
>chr10:122448339+122448466 128bp CGCTGCATGACTTTAAATTGG CAGGGGTGGAGCTGAGAAT
Mut= 129 bp
Wt= 128 bp
Fam=Mut
Hex=Wt
Sequence
Wt Sequence (deletion in lower case):
GGGCCTGGGGGCTTGGAAGGGTGGACCCCAAATCCAGGCGCACTGAGTTCGCAGCAGCAGCAGACGCGGATCTCTACGTACGCTTTTACTACACCTGTTGACGCGCTGCATGACTTTAAATTGGCTTATAAAGGAGGCTGGAGAAAttccgaggagcccagagaagtttggggcagatccggaaagcgcccggctctgcagaggaatctaggattctcagctccacccctgcagcaccaaagagaaaaggacaaatcaccaacgacttggaggggttgggtgtaggggacagtctccagccactaagatgagaaggagcgcatcctgaggcagcctaagactgctttgggagcagggagagtccgctcccttgcctcggacaagccggagcagggacacgcaagaacatcccatccaaaaccactctgagcgcgcgtgggattgcgcagcgaagagagcgaggtaaggaaggacggtgcgcagaacttaggacgcgggtcttcgggtcctccgtggagaagaggaagcagcccacgaggaacagggttccccggctctgtgccgacagcatgagtttcccgcgaggctcccacgatctgcccgcgggcaactccagcccgtggtggcctctgaccaccgagggcgccaacagcagccgggaagcagctgggctcggggaaggcggcagcccgccgggggacgtacgcaacgaggagctggcgaagctggaggttaccgtgctggcggtgattttcgtggtggccgtgctgggtaatagcagtgtgctgctggcgctgcatcgcacgccacgcaagacatcccgcatgcatctcttcatccgacacctcagcctggcagacctggcggtcgccttcttccaagtgttaccacagctgtgctgggacatcacctaccgcttccgcgggccggactggctgtgccgggtggtgaagcacttgcaggtgtttgccatgttcgcatcttcctacatgctggtggtgatgacggctgaccgctacatcgccgtgtgccacccgctcaagaccctgcagcagcccgcgcgccgctcgcgcctcatgatcgccgcctcttgggggctgagtttcgttctgagcataccacagtactttatcttctctgtgatcgaatttgaggtgaacaatggcaccaaagcccaagattgctgggctaccttcatcccgccctggggtacccgtgcctacgtgacctggatgaccagcggggtcttcgtggtacccgtgatcatcttgggtacctgctatggcttcatctgttaccacatctggcgcaatgtccgagggaagacagcatcgcgacagagcaaaggcggcaagggctctggggaagccgcgggtcccttccacaaggggcttctggtcacgccttgtgtcagcagcgtgaagagcatttcccgtgccaagatccgcacagtgaagatgacctttgtgattgtaagcgcctacatcctctgctggacacctttcttcatcgtccagatgtggtcagtctgggataccaatttcgtttggaccggtatgtactgagactactatggaagaggtgtgtgtgtgtgtgtgtgtgtgtgtgtgtgcctgtgtgtttgtggtgtgtgttggggggggagtgtttgtggtgtgtgtgtgtatgtatgttatttttcatacaacctattcaaTAGTTAAGCTTCTATGTAAGAAGTCTTAAATGAACAAAATTGCAAATTCCTGGGACCTCCACCCCAGGTACACTGGAGCCCAGGATTCAGCATCTGCAGCATGTGTGACCACCCTAAGGCCACCAGCTGTGCCTAGAGTCACTAGTTACAAAACACTTGTGACATTTTGCCAAATTAC
This mutation is a 1550 bp deletion beginning at Chromosome 10 position 122448382 bp and ending after 122449931 bp (GRCm38/mm10).
JAX Protocol
Protocol Primers
| Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
|---|---|---|---|---|---|---|
| 43365 | CGC TGC ATG ACT TTA AAT TGG | Common | A | |||
| 43366 | CAG GGG TGG AGC TGA GAA T | Wild type Reverse | A | |||
| 43367 | ATC CTG GGC TCC AGT GTA CC | Mutant Reverse | A | |||
| 43368 | Fluorophore-1 | CGA GGA GCC CAG AGA AGT TT | Quencher-1 | WT Probe | ||
| 43369 | Fluorophore-2 | CAA ATT CCT GGG ACC TCC A | Quencher-2 | MUT Probe |
Reaction A
| Component | Final Concentration |
|---|---|
| Kapa Probe Fast QPCR | 1.00 X |
| ddH2O | |
| 43365 | 0.40 uM |
| 43366 | 0.40 uM |
| 43367 | 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.