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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
>chr3:90543152+90543251 100bp CTCTTGGCTGCTTCCTTTGT GCAAGTCCCTACAGCCTCAG
Mutant= 97 bp
Wild Type = 100 bp
Sequence
AGCCCCTGCTTTCCTCTGTCTTGATTCTTCCCAGCTAAGGTTGCCACAGAACAAGGGACCTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTAGGGGGCAACAGTTACCTGCTTCCTCTCCTCACAGGAGCCTCCTGCTCTCAGAGGGCTCTGACAGGCATCAGGCCTGCCTGGGGCAGGGGAAGGGCCTGGAGAACCTGTCTTGgcagttggcagtctggctggggaacacctggattagaggagtcacatagctctccttccaggccacccccacacccactttgctatcctatgtctgctttctcccttctctgaaaggcaagaagtctgaggatgctgtctgtgtgcagggatgtcccttctcatctctcttttctgcttcctaggcccagagaggagctgaggcagtgagatggctgactgctatacagagctggagaaggcagttgttgtcctggtggaaaacttttataaatatgtatccaagcacagcctggtcaaaaacaagatcagcaagtccagcttccgaaagatgctccagagagagttgaaccacatgctgacggtatccccactgctgccctctgtgcctgctcttctcctcacagctctctcagtgacccctccctccctgtgctctgctccgtgttccctcctagcctgctagcctcctctgccctgctagcctggccaagggactctgcctagggtcctgtctgggccatcccaagctttactgattcaagttcttccacaggacacagggaaccgaaaggcagctgacaagctcatccagaacctggatgccaaccacgacgggcgcatctgctttgacgaatactggaccatgattggtggaatcaccagccccatggccaacctcatccgccaacaggagtgccagcaggagagccagcaggagtgccagcaggagagccagcaggagagccagcaggagagccagcagggcagcagctagaggcccctcgtgggccccttttcatgtcctgacccatgtcctgccagtgttctttccatgctttaagttgactctttcttcctcccttctccctccagacctgttcctacccttgctgaacagcacaagatgggtgggtcgggggctatcaaggccactgtggtagtgcttccctgagcattcaatccctggatcctgtaggccctgggagggggtcccctttgtgaaatatcaatgctgtttggggccctccccccaagacttctctcacttgttgagtctgtgtgctctcgcaatgttcctgtctaattcttgactgattctgggtctgtcctgacctctagaggtgagcttgctgcttgaggtctcccagctcctgactgtggcagctgtggtagtggcagtaccagaattagccgcttcttttcctcccaacctcttagccgggtagagaggcattcccaggggctcctgtccagctgtctctgtctgggaatgatctaacctgggttgagcctcctctctgctctgccaccgaaataaacgttgaatttggagtttgtaactcttggctgcttcctttgtctttaattgctccacacttcttgggaatctattctaccttggtgATCTTTGTCCTGGACCCTGAGGCTGTAGGGACTTGCATAAGAAAACATTACATGCAAACAAAATAAGAAGAAGTTAAACTGGGATTGATGGCCCGCATGTATAATTCTTGAATCTAGGCTGATGTATTAGGGGTCTGGAGTTCAAGGCCAGCCAGAGATGCATAGCTCAGCATGGTGGCGTTTGCCTGCCCAAAGGCAAAAAGAATAGGTTATATTCTCAGATTCAAGTTCAACTTTTAGGTGCATAGTAGGTGT
A 1406 bp deletion beginning at Chromosome 3 position 90,541,810 bp and ending after 90,543,215 bp (GRCm38/mm10).
JAX Protocol
Protocol Primers
| Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
|---|---|---|---|---|---|---|
| 45960 | AGG GCT CTG ACA GGC ATC AG | Mutant Forward | A | |||
| 45961 | CTC TTG GCT GCT TCC TTT GT | Wild type Forward | A | |||
| 45962 | GCA AGT CCC TAC AGC CTC AG | Common | A | |||
| 45963 | Fluorophore-1 | TGC TCC ACA CTT CTT GGG A | Quencher-1 | WT Probe | ||
| 45964 | Fluorophore-2 | AGA ACC TGT CTT GAT CTT TGT CCT | Quencher-2 | MUT Probe |
Reaction A
| Component | Final Concentration |
|---|---|
| Kapa Probe Fast QPCR | 1.00 X |
| ddH2O | |
| 45960 | 0.40 uM |
| 45961 | 0.40 uM |
| 45962 | 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.