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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
>chr11:84473300-84473398 99bp GGCCACTGAAGTCTTTCTAGC CTCCAGTGCCTGTCTCATGT
Mutant= 90 bp
Wild Type = 99 bp
Sequence
Wt Sequence (deletions in lower case):
AGGAGAGAAAGAACTGAGCTCATTCTTCAGAAGAGGGGAGGGCCTTTCAGGAGGAATTGCTTATCAGACTCAGCACCCTTGAGACCTGAGGGCAGCCTGTAGGATATACCCGAGGAACTTAATATCAGGGGCAGAGTTCATTATGGTAACAGGCAAGGAGTTTGCCAAACTTAATGACTTTAAGAACTGGCCACTGAAGTCTTTCTAGCTGCAGACACATTTCTAAGCACCTTCCACgtgctgctatcttgcttattccttagcgtcacatgagacaggcactggagtagtttccatggagttgatctcagaaagtactgactgtccccactcagaacttgtcaggagacagggagtgacagcaattggagagttgtaaatgactaattatcccttttctctgctttatttctattccaggtcacaaagcacttaaagccttgttaaggtcattggtggatcttcaggaagagttgcttttccagtacccagacacgagacatatagtaaatggggcgaaacccaatacagagaggtaaggaggaagcagagccatggcttgattattttcttctcatctgaatgaaaaaggaaggcgggtctttctcagacagatagatacgggatttgataaacgtaaaatagagtcgcttttatatttaacAGGTGGTCAGAACAGGACAGTTTAGAGTGCTTGTTCTAGTTTTTTTTTGTAACTGCTTATCTTTATAGCTTAAGGCAAAATATTTGCCTTCTCTTTTGTTCTATAATAGTGTACTGGCTAGTTTTGTGTCAACTTGACACAGGCTGGAGTTATCACAGAGAAAGGAGCTTCAGTTGAGGAAATGCCTCCATGGGATCCAACTGTAAGGCATTTTCTCAATTAGTGATCAAGGGGGAAAGGCCCCTTGTGG
A 427 bp deletion beginning at Chromosome 11 position 84,472,923 bp and ending after 84,473,349 bp (GRCm38/mm10).
JAX Protocol
Protocol Primers
| Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
|---|---|---|---|---|---|---|
| 49582 | GGC CAC TGA AGT CTT TCT AGC | Common | A | |||
| 49583 | CTC CAG TGC CTG TCT CAT GT | Wild type Reverse | A | |||
| 49584 | AAC TAG AAC AAG CAC TCT AAA CTG TCC | Mutant Reverse | A | |||
| 49585 | Fluorophore-1 | CAC CTT CCA CAG GTG GTC AG | Quencher-1 | MUT Probe | ||
| 51370 | Fluorophore-2 | TGC TGC TAT CTT GCT TAT TCC TTA G | Quencher-2 | WT Probe |
Reaction A
| Component | Final Concentration |
|---|---|
| Kapa Probe Fast QPCR | 1.00 X |
| ddH2O | |
| 49582 | 0.40 uM |
| 49583 | 0.40 uM |
| 49584 | 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.