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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
>chr11:55181709-55181797 89bp GAGACCCAGATGTGCCAAAG CGTGCCAGCACTTCAATTAC
Mut= 92 bp
Wt= 89 bp
Fam=Mut
Hex=Wt
Sequence
Wt Sequence: (deleted region in lower case)
ATAGACAAATTATCTAAGTATTTTGGGGCCTCCATTTCCCATCTACAGACCAAACCCCAACCATTGCCTACTTTATTGTCCTGCCAAGACGATTAAATGAGATGTTATATTTAAAGACTCCACGCATACAGGGTCTCATACAATTAGTGTTTCATGGTACAGACAGGGCCCAAGCCGCTAATGGAACAATTCTGGATATCTTGGGATGGGTGGTCACCTTCCAGGAGACCCAGATGTGCCAAAGCAAGGATACAGGAAAAtcgagatggccatgaatttacctgcccacgcaggtaattgaagtgctggcacgctatgaggggatggctgctaggtcctttcccagctccatttccatgtcctctgactgccttcctttcctgtagcgggttccagacattggttcatctggtcaaaggcaacatgggcacagggatcctcggacttcccctggctgtgaagaatgcgggcatcttggtaagaggcatggctaagtgggggcactacgaagaagctgtgggatagggaagaagacataatggtttaaaataccttgtagcagtcagtgcttggggggtggagaggtatctgacccagacataaaatctgaacatcaagtctgagtctgagttctgccttggcttggcctctggcattaggagtcaagtcaacactttttatccaccctcaggcttcaaatccctccttttgtagaatgtaaggattagaggaggaccagTTACCCAGTCTACCTTTACAGATGCCCTTGGGGACATCTCAGAGACCACCAATGGGCCATGTGATCAGGACCCTGAGTCTCCTGCTCCATCACAAATGTCACCGTGCTTGCTATGTGTACTTTTTAGACATGACTTGAGGTTAACTTTCGGAAAGAGTAGCCAAGTCTCTTTCAACTTGGTTTTCTAGAAAGTTCTTTCAGGACTAGCAAATGGCTCACGAGATAAAGGCACTGGCCACCAAGTC
This mutation is a 479 bp deletion beginning at Chromosome 11 negative strand position 55,181,761 bp and ending after 55,181,283 bp (GRCm38/mm10).
JAX Protocol
Protocol Primers
| Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
|---|---|---|---|---|---|---|
| 38476 | GAG ACC CAG ATG TGC CAA AG | Common | A | |||
| 38477 | CGT GCC AGC ACT TCA ATT AC | Wild type Reverse | A | |||
| 38478 | CCC ATT GGT GGT CTC TGA | Mutant Reverse | A | |||
| 38479 | Fluorophore-1 | AAT TTA CCT GCC CAC GCA | Quencher-1 | WT Probe | ||
| 38480 | Fluorophore-2 | CAG ATG CCC TTG GGG AC | Quencher-2 | MUT Probe |
Reaction A
| Component | Final Concentration |
|---|---|
| Kapa Probe Fast QPCR | 1.00 X |
| ddH2O | |
| 38476 | 0.40 uM |
| 38477 | 0.40 uM |
| 38478 | 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.