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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
>chr8:78246026+78246110 85bp AGCCTGTCTGGTGTTTAGAGAT GAGTTCCTGTGGCTGGGAAC
Mut= 83 bp
Wt= 85 bp
Fam=Mut
Hex=Wt
Sequence
Wt Sequence (deletions in lower case):
AGAAATTAATGGACTTTTAATATTCAACAAGTTTTTTTTCTTGTTGGTCTTCTTACTTGGCTGGCAGGTCTGTTTGGTCTGCTGATAGGTTGGTTGGTTTGAGCAGGGGTGTGTGTAGAGGGTAGGGTAGGGAGTGGACGTGTTTGTATTTGTCTGTGTGTATGAAAAAAAAACTTTGAATAGTTAAAGTAACACATTTGTCCTTTTCAGAGGTGCGGTTACATTAACACAACTGTGTTAGGTAAGCTCTCTGCATCCTTCTAAAATGGAAGTTGCTTGATTTGTCACAAAGATCTCTTGATAGTTTTCATGCTTTCGCTGAGGAAGATGAGTAAGAAAGCCTGTCTGGTGTTTAGAGATTGAGAACCcctctctggtctaaaatggctggtgttctgctcgtgttcccagccacaggaactcatacaagaagagcatctgcgtggacatgctgcgagatgggttccacaagtccttcaccgagctctttgcactgatggaacagtgggataagctccgggaagctgcgaaggcacagtccctcttctggcagcagaggcctctggaggaccagccggacaagctagacaatttctaccactacctgaccagggccgaggctgccgagaggaaaggtaggtgctgggatgtggggttgcaccccagggaagattccaaaatgtcaccctatgaatttgatacattaagacaaatcacacagttcaacctattccactgagtggctttaaggCTAAATGACCAGCTACAAAGACATTCTGCAGATGTCATTGCAAGCCTGGGAAGACAGTGTTCTACCAAACATTTCCATAAGTTTCCAACAGGTGTAAACCTGTCCTATCATTGAGAACACATCCTGATTTGTCTTTGTGTTGTATGAATATTGACCAGTTTTCAAGGCACTGTCACATAAAAACCACACAGACATAGTTTGTTTTAGCATCTATGTAACGAGCGTTCAAAGTTTTGTCATGAGATCCTGTCAGCTTGGATTAGAAAATGGTAGTATTTAAAAAGACATGAAAAGCCTGTAATTTCTAGGGTATTGTTAAGGCTGTAATCCCAGCACTTGGGACAAAGAGACAGCTTATTCTGAGTTTGGGGTTAGCCTCAGCTACAT
This mutation is a 383 bp deletion beginning at Chromosome 8 position 78,246,056 bp and ending after 78,246,438 bp (GRCm38/mm10).
JAX Protocol
Protocol Primers
| Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
|---|---|---|---|---|---|---|
| 48399 | AGC CTG TCT GGT GTT TAG AGA T | Common | A | |||
| 48400 | GAG TTC CTG TGG CTG GGA AC | Wild type Reverse | A | |||
| 48401 | CTT CCC AGG CTT GCA ATG A | Mutant Reverse | A | |||
| 48403 | Fluorophore-1 | ACC CTA AAT GAC CAG CTA CAA AGA | Quencher-1 | MUT Probe | ||
| 48406 | Fluorophore-2 | CTG GTC TAA AAT GGC TGG TGT T | Quencher-2 | WT Probe |
Reaction A
| Component | Final Concentration |
|---|---|
| Kapa Probe Fast QPCR | 1.00 X |
| ddH2O | |
| 48399 | 0.40 uM |
| 48400 | 0.40 uM |
| 48401 | 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.