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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
>chr13:3933373+3933515 143bp GTTGAGCAACACCACAGCA GATCTTCCCTGGCCTCTACA
Mut= 140 bp
Wt= 143 bp
Fam=Mut
Hex=Wt
Sequence
Wt Sequence (deletion in lower case):
TCCTCCCTCCCTCCTCTCTCTTTCTGAAGATCAAACTCAGACCTGCATGCTTGTAAGGCAAGCACTTTACTGATGGAGCCTTTGCCCTAGCCTCCTTTCTGAGTCCTTTGTTCTTGGGAAGCTATGTTCTACCATTATCATGGTAAACAAACAAGAAGGCACTTTCAGAGTTTGGATATGAGTATTCTTCAAGTAAGAAAGTACTGATTCCTTCTAGTTAACTGACTGTAATTCTCACTCCCCCCCCCCCGCCCCCAGgcagaacaatgcagtggacttcagggatttttgatttaccgaagctttggaggaggcacagggtcgggatttacatctctcttgatggagcgactctccgtagagtattgcaagaagataaaattggagttctctgtctacccatctcctaggatctccactgccgtggtagaaccgtacaatgccatcctcaccactcactccaccatagaatactcagattgtgctttcatggtggacaatgaggctctgtacgacatctgccagcacaaacttggcattgaacgtccttcctatgctagtatcaacaggctgattgctcaggtgtcgtcttccatcactgcatcactgcgttttgaaggacctttgaatgtagacttaattgaattccagaccaacctagtaccttatccaagaatccatttccccatcacagctcttgcccccatcatctccgctgaaaaggcgtaccaagagcagctctctgtgtctgacgtcactgcttcttgctttgaggtctccaaccagttggtcaagtgtgatcccaggcttgggaagtacatggcttgctgcttactgtacagaggagatgtggtccctaaggatgtaaatgaagcaattgcagccatgaaatctaggacctctgttcagtttgttgattggtgtccaactggtttcaaggtgggcatcaattatcagccacctgcagtggtaccaggaggggatctagctagggttcagcgtgctgtgtgcatgttgagcaacaccacagcaattgtggaagcctgggcccgcctggaccacaaatttgacctcatgtatgccaagaaggcattcctgcactggtacatcacagaagGCATGGAGCTTGGGGAGTTTGTAGAGGCCAGGGAAGATCTGGCTGCTCTGGAAAAAGATTATGAAGAAGTGGGTCTGAGTTTCTGACTCTTTGTAAGTCTGGTTTTTCCTTTATTCATGTGTCTATGTGAGGTGTGTGTGCTTATTTGCATATGTGCACTCATATGTGGGGAAGCACGTGTATGCACAAATGTATGTGCCCATGTGTGCACAAGTGTGTGGAGGGCAGATGCTGAACTGGGTGTGTGCCTTCCTT
This mutation is a 859 bp deletion beginning at Chromosome 13 position 3,932,618 bp and ending after 3,933,476 bp (GRCm38/mm10).
JAX Protocol
Protocol Primers
| Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
|---|---|---|---|---|---|---|
| 49005 | GTT GAG CAA CAC CAC AGC A | Wild type Forward | A | |||
| 49006 | GAT CTT CCC TGG CCT CTA CA | Common | A | |||
| 49007 | GGC ACT TTC AGA GTT TGG ATA TG | Mutant Forward | A | |||
| 49008 | Fluorophore-1 | CCT GGA CCA CAA ATT TGA CCT | Quencher-1 | WT Probe | ||
| 49009 | Fluorophore-2 | CTG ACT GTA ATT CTC ACT CCC CC | Quencher-2 | MUT Probe |
Reaction A
| Component | Final Concentration |
|---|---|
| Kapa Probe Fast QPCR | 1.00 X |
| ddH2O | |
| 49005 | 0.40 uM |
| 49006 | 0.40 uM |
| 49007 | 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.