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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
>chr13:56450547+56450694 148bp CCCCAAGACCATGAACTCAG TCCCAACACATCTCCTCACT
Mut= 146 bp
Wt= 148 bp
Fam=Mut
Hex=Wt
Sequence
Wt Sequence (deletion in lower case):
TCCCTTTGTCAGCCTGTCACCACAGACTTAGTGAGTTGCACAGTGTTGCCTGTCAGTTTGTCTAAACCCTCCATCCTGTCCTCATCTGCCTTACCCCCAAGACCATGAACTCAGTGAAGGTGAACACTGTTTTGTTATTGATTTGACTCATAGAAAGTAATGAGagagacagacagacagacagacagacagacagacagtgctagctttgcgtgagcagctagtgaggagatgtgttgggacttaacatccccatgactcttgagggctcctcagacattctgcagccacagttctgacactgccatttgggacatgcatatatctgaggccaagactccattgtgcaggaggctcatctttggggctagaatgactgcctgagttgcatgtggccttctcctatcatgccatgtcatctagacccttgctgggggctcttctgtggcatggtggattattaactctgagcctggggagcagggaggtcccatacttggtatgcagagagagaccctgaagagccgtttggcctcacacttcctcccattgggtctccttgccacaggtgtttggcttcttcaagggcatgtccttccccctggccagcatcgccatctataactcggtggtgttcggggtcttcagcaacacccaacggttcctcagcaagtaccgctgtggggaactcgaggctggccctggccgaagtctgtctgatctgctcctggctagcatgctgactggtgtggtctctgttgggctgggcgggcccgtggaactcatcaagatccgactgcagatgcaaacacagccatttcgggaaggtaagaggccgatggagagaaatctggtgtctgaggacccgtgaccctttcccatttgtctggatctgtggtcccattgttcataaccttctctgtgagcaagaccaccctcttccctgctcaccacagggagcatctcattttctcctcttgggagtttcccatgctgcttttgaaggtggctttgacagaggaggaaagagaggagaggggaacccgctgtgtgtcacctttggctgacttgagtgctggaggagacatatttcgattctgtgggttttgttcttggttcagtctcaagtctccatgtctatagaatggaggggctggatctctggtctttcctgtcatagTGTATGTATTATGTATGATGAGTCCCCCCAACCACCCCGCCCTTGCTGGCAGCCTTCAGAATGGTCACTGTGTGAACCCTCTGCACTCTAAGGCTGGGGTTCAGCTGTGGAACATGACAGGAGGCCCACAAGCTCACTCAGGAGCATCCTGGCC
This mutation is a 1016 bp deletion beginning at Chromosome 13 position 56,450,617 bp and ending after 56,451,632 bp (GRCm38/mm10).
JAX Protocol
Protocol Primers
| Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
|---|---|---|---|---|---|---|
| 45651 | CCC CAA GAC CAT GAA CTC AG | Common | A | |||
| 45652 | TCC CAA CAC ATC TCC TCA CT | Wild type Reverse | A | |||
| 45653 | TTC ACA CAG TGA CCA TTC TGA AG | Mutant Reverse | A | |||
| 45654 | Fluorophore-1 | ACA GAC AGT GCT AGC TTT GCG | Quencher-1 | WT Probe | ||
| 45655 | Fluorophore-2 | AGT GTA TGT ATT ATG TAT GAT GAG TCC CC | Quencher-2 | MUT Probe |
Reaction A
| Component | Final Concentration |
|---|---|
| Kapa Probe Fast QPCR | 1.00 X |
| ddH2O | |
| 45651 | 0.40 uM |
| 45652 | 0.40 uM |
| 45653 | 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.