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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
>chr3:98427142+98427246 105bp TGCCAACTTGTCTGTTGCAG GGGAACAGAGCCATGTCAG
Mut = 112 bp
Wt = 105 bp
Fam = Mut
Hex = Wt
Sequence
Wt Sequence (deletions in lower case):
GGTTGAGAATCCCCCCTCCCACCCCCGCCTCGTTTTTTTCTTCTGGAAACATATTCCCATCTGGTACAAACCTAGCCATTCGCAGGCTTCAGGTGATTCAGCTGAGCAGAAGCTGGGGTGTCTAGTAGGCGGTGTCTGCCAGGAGAGTTGGCAGTGGGGCAGAGTGCATTATCCTTTAGTCTTAGCAGGAGGAAACTGGTAGGGAAGAGGCCGAGTAAAAAGTAGGGGTAAGGGGGCTGGGGGAGGGGCATTACTCCTTTGGCTCACCCTCCTGGCACATCAGGTCTAGTTGACCCCATTGCCAACTTGTCTGTTGCAGAGGGGCGGCTGAGTgaggaagaaagggctatcctccgtgaggaagaggacgaccagcctggtgtggctgacatggctctgttcccaggactgtcagaatctgacagcatatcccggagtccgcggggagaggaagatgaagaggaggaagacgaagaagaggaaagcgctggggagaaccgcttgatagaggaggaagatccattgcctactcccgtgcttccttggaggcgtcacctctccctgggcggtcgacaccggggtgacaagcctgctcaccgccgcttccaccggctccatcaccccatggccatggacctcggggaactagacagcctgatggccagtatcatggacgcgcccaccatctgcccagactgcggggagagctttagcccgggcgccgcattcctgcagcaccagcgcatccaccgcctggcagaggccgccgccgtggccagcttggagcccttcggtttggccggcgagtgcggaggagtggtggggatgatgggcatgggtatgggcgtgggcatgggcgtggccggcggcttcggggcggggcccacgctggcccggccccctcgcgaaaagcccttccgctgcggggagtgcggcaaaggcttcagccgcaacacctacctgaccaaccacctgcggctgcacacgggcgagcggcccaacctgtgcgccgactgcggcaagagcttcagctggcgcgccgacctgctcaagcaccggcgcctgcacacgggcgagaagccctacccgtgcccggagtgcggcgaagccttcagcctcagctcgcacttgctcagccaccggcgcgcgcacgcggcggcgggcggcggcgcgggctcggcgggctcggcggcggcgctgcggcccttcgcatgcggggagtgcggcaaggggttcgtgcgccgctcgcatctggccaatcaccagcgcatccacaccggcgagaagccgcacggctgcggcgagtgcggcaagcgcttcagctggcgctcggacctggtgaagcaccagcgcgtgcacacgggcgagaagccctacatgtgctccgagtgcggcgagaccttcagcgtgagctcgcacctcttcacgcacaagcgcacgcactcgggcgagcggccctacgtgtgccgggagtgtggcaagggcttcggccgcaactcgcacctcgtcaaccacctgcgcgtgcacaccggcgagaagcccttcggctgcggccagtgcgagaagcgcttcagcgacttctccacgctcacgcagcaccagcgcacgcacaccggcgagaagccctacacatgtctcgagtgcggcaagagcttcatccagagctcccacctcatccgccaccggcgcatccacaccggcaaCAAGCCTCACAAATGCGCGGGCTGTGGCAAGGGCTTCCGCTACAAGACGCACCTTGCACAGCACCAGAAGTTGCACTTGTGTTAGGGAGGGCATCCGAATGAGGCGGCGGTGGGAGTCGTCTGGCTACTGATCTGTAGATGTTGGGGAAGGGGCAGGAGGGACAGTCTGAGAGTGTCTGGGGGAGCCTTTTGGTGTTTGCCACCCCACCCCCCAGGAGATGGTTAATAAGAGTTGTTACTTCGAGGTGCCTACCTCCACCTTCAAAGTAACTCTTAGAAGA
This mutation is a 1360 bp deletion beginning at Chromosome 3 position 98,427,181 bp and ending after 98,428,540 bp (GRCm38/mm10).
JAX Protocol
Protocol Primers
| Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
|---|---|---|---|---|---|---|
| 49298 | TGC CAA CTT GTC TGT TGC AG | Common | A | |||
| 49299 | GGG AAC AGA GCC ATG TCA G | Wild type Reverse | A | |||
| 49300 | AAG TGC AAC TTC TGG TGC TG | Mutant Reverse | A | |||
| 49301 | Fluorophore-1 | AGG GCT ATC CTC CGT GAG GAA | Quencher-1 | WT Probe | ||
| 49302 | Fluorophore-2 | AGT CAA GCC TCA CAA ATG CGC | Quencher-2 | MUT Probe |
Reaction A
| Component | Final Concentration |
|---|---|
| Kapa Probe Fast QPCR | 1.00 X |
| ddH2O | |
| 49298 | 0.40 uM |
| 49299 | 0.40 uM |
| 49300 | 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.