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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
>chr11:110072010-110072095 86bp AAGGAAGCAGCCTTTCCTCT GCTTCACTTAGCTGTGGTTAGACA
Mut= 80 bp
Wt= 86 bp
Fam=Mut
Hex=Wt
Sequence
Wt Sequence (deletion in lower case):
TTAGTATTACTCCTTTCAGAGTTCTAAGAAATAGCTATATTTGTTGGCTAGTTAATGTTTAGACTCCAATGACGGCTAGCACCTCTGTTCATGGCATTTCTTCTTGTTGCCCTTCGATACTGCCTGGCTGACCTGAAGAGATTCTGGACTTTATGCAGatgttttttctaacatctgtaaagtgttagtttcctactttgccttgctatggtagctgtccacattgtatcattttctagcttaccctatcactacgtatggttcagctcaggaaaaatgctcacagtggcttgaacagtgtccaggtctcagtccacaccaaaggaagcagcctttcctctaggtaagatcaggctttaaaacactggtccaagtaccattgctgtctaaccacagctaagtgaagctcacatgccatcccttatatccttgcaagccaaatctgcgtaaagatttcagccagactaggagttagatagtaaagaaaaccattgctccttaaacagtttatctcttgctctctttctcagaatcagaaatcttacaaaagactacatacaaaagtctaagcgcacagaagctttgaaaggtaagtaactaaggtagaaaaatatggtttttagtcagttaacttttctctccatgagcaattctagactgtgtaagtggatgttgagtctcctgaccccccaaaccccacaactgagctaacattgacaggctgtgtgattttgagtattaactttgagtattaattttgatttaataagtgaatgaatgtatacttttataaattctagtcacactataaatacgtaggcactatatgagccctgaattaaacctacatttgtgattttataccttcatgctgttttcctcccagatctgacactggatgtatataaaggccagattactgcgatccttggtcacagtggagctgggaaatcaacactgttaaatgtcctgagtgggttgtgtgttcccaccaaaggtaagagacagagtctttcctctaagttggcaatcatctgtgcttttgttagacatttgagcattaaggtcagacagagataaactcccagctattgacacataatctctattactcttttataaaatgtatatgatattaactgaaatgtacagaattttagtattttagaatactcaaaattttatagaattaacagtttcaattgatattgatcacatttttctgtatagataaagtaaccttgaaaattagcaacttttaaaatatgACTGGAAAGGTCTCTCAATCTCACTGTTTTGACTGTAACTTATAACCTAGAATGGCTTTACCTACATGTGTGAGACTTTAGCTGGGCTATTCTCCCCTTCCCCTTCTCCCCTCCCTTCCCTCTCTATCTCCTTCACCCTCTCTATCTCCTTCATCCCCTCCCTCTCCTTCTCCCTCTTCCCTTTCTCC
This mutation is a 1110 bp deletion beginning at Chromosome 11 position 110,071,149 bp and ending after 110,072,258 bp (GRCm38/mm10).
JAX Protocol
Protocol Primers
| Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
|---|---|---|---|---|---|---|
| 46922 | AAG GAA GCA GCC TTT CCT CT | Wild type Forward | A | |||
| 46923 | GCT TCA CTT AGC TGT GGT TAG ACA | Wild type Reverse | A | |||
| 46924 | GAT ACT GCC TGG CTG ACC TG | Mutant Forward | A | |||
| 46925 | TAC AGT CAA AAC AGT GAG ATT GAG AG | Mutant Reverse | A | |||
| 46926 | Fluorophore-1 | TCA GGC TTT AAA ACA CTG GTC C | Quencher-1 | WT Probe | ||
| 46927 | Fluorophore-2 | CTG GAC TTT ATG CAG ACT GGA AA | Quencher-2 | MUT Probe |
Reaction A
| Component | Final Concentration |
|---|---|
| Kapa Probe Fast QPCR | 1.00 X |
| ddH2O | |
| 46922 | 0.40 uM |
| 46923 | 0.40 uM |
| 46924 | 0.40 uM |
| 46925 | 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.