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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= 119 bp
Wild Type = 136 bp
>chr13:111255114+111255249 136bp AAGATCCCAGCCAGCAACC TGGACGACCAACCATGGAG |
Sequence
Wt Sequence (deletions in lower case):
AAGATCCCAGCCAGCAACCATGGTTGACGATGAGCTGACTGCTTTGGtagtagataatgggtcagggatgtgcaaggcaggttttgggggtgacgatgccccccgtgctgtgttcccctccatggttggtcgtccacgacaccagggggtcatggtaggcatgggccagaaggactgttatgtgggagatgaggcccagagcaagcgaggcatcttgaccttgaagtatcccattgaacacggagtcgtcaccaactgggatgatatggagaagatctggtaccacactttctacaatgagctccgggtagcaccggatgaacatcctattcttctcaccgaggcaccccttaaccccaaaatcaaccgggaaaagatgactcagataatgttcgaggctttcaacacacctgccatgtatgtagcaatccaagccgtgctatccctctatgcttccggaaggactacaggtatcgtgatggattctggggatggggtcactcacactgtgcctatctatgaaggttacgctctgcctcatgccattctacgactagatctggcaggtagggacctgactgattacctcatgaagattctgacagaacgaggctataacttcaccaccacagcagagagggagatcgtgcgggatgtcaaagaaaagttgtgctacgtggccctggactttgagcaggagatggtcactgcagccgcttcatcctcactagagagaagctatgagctccctgatgggcaggtgatcaccattggaaatgaacgcttccggtgcccagaagccattttccagccttcctttctaggcattgaatccagaggcatccatgagacaaccttcaactcgatcatgaagtgtgatgtagatattcgtaaggatttatttgccaacactgtgctgtctggaggcagtaccatgtacccaggcattgccgacaggatgcagaaggaaattgtaaccttggcaccaagtaccatgaaaattaagatcatagctcccccagaacgcaagtattcggtttggattgggggctccatcctcgccagcctgtccacattccagcagatgtggatcagtaagcaggaatatgacgaagctggacctcccattgttcatagaaaatgctTCTAGGTAAGCTACCGTGTCCCTTTTCCTGGGTCATAATGATGGTACCCCACCTCACCCAGGACACTGAAGG
This mutation is a 1098 bp deletion beginning at Chromosome 13 position 111,255,161 bp and ending after 111,256,258 bp (GRCm38/mm10).
JAX Protocol
Protocol Primers
| Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
|---|---|---|---|---|---|---|
| 66035 | AAG ATC CCA GCC AGC AAC C | Common | A | |||
| 66036 | TGG ACG ACC AAC CAT GGA G | Wild type Reverse | A | |||
| 66037 | CCT TCA GTG TCC TGG GTG A | Mutant Reverse | A | |||
| 66038 | Fluorophore-1 | AGT AGA TAA TGG GTC AGG GAT GTG | Quencher-1 | WT Probe | ||
| 66039 | Fluorophore-2 | CTG ACT GCT TTG GTC TAG GTA AGC TA | Quencher-2 | MUT Probe |
Reaction A
| Component | Final Concentration |
|---|---|
| Kapa Probe Fast QPCR | 1.00 X |
| ddH2O | |
| 66035 | 0.40 uM |
| 66036 | 0.40 uM |
| 66037 | 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.