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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
>chr14:75291907+75292051 145bp TACAAGGAAGGGTTAAGGTTTG GAAAAACCACAGGAAGGTGTGT
Mut= 150 bp
Wt= 145 bp
Fam=Mut
Hex=Wt
Sequence
Wt Sequence (deletion in lower case):
GGGTTTGGCTTAGCAAATTTTATTTTATAAAAGGAAATACAAGGAAGGGTTAAGGTTTGTAAAGTTCTCATTATATAATATTTTTGTATGTTCAATGTATACGATGTATTTCGAGCATATTAATACCCCattccaggaactactctctacctaaacacacacacaccttcctgtggtttttcattcccttatggtgttattattattggagtggcatatgcttagctggcatttgggtctctctgacttgattttgccctggtaccatcttttctccagacatttacatagtaatatttgcctcatagacacagtgtcgtaactggctgaagactggcagctgcctctatggaaacacatgtaggttcatacatggcccttcacctcgtgggaaaggctacagcagcaattacagaaggtaaactgagaactcagatattttctatagtattaaaaaccaacttttgtagtttagtgtaaaaatatgaaataaaaccatatatttggtcagattttctttttttggatagtcttattttggttgttactgttaagattaataaaactttagctttaaagtgtacatgtttacatgttttggtagtagatccatttttcaatgtgtatattcaaatcaatgaacacctgtggagagactgagtctgggaaagaacagcccttaaagagcccctACCTGACACAGCCCCAGGTGTGTATTAAATTTCTGCTTAGGCACAACTCAGACGTTTGAGAGAGTTTACTTTTCGTAAACGAGTAAAATTTAAATAAGGTTTAATTGTAATTTTCTACAGGCATTTTAAATTCATATCT
This mutation is a 563 bp deletion beginning at Chromosome 14 position 75,291,999 bp and ending after 75,292,561 bp (GRCm38/mm10).
JAX Protocol
Protocol Primers
| Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
|---|---|---|---|---|---|---|
| 41660 | TAC AAG GAA GGG TTA AGG TTT G | Common | A | |||
| 41661 | GAA AAA CCA CAG GAA GGT GTG T | Wild type Reverse | A | |||
| 41662 | CAA ACG TCT GAG TTG TGC CTA | Mutant Reverse | A | |||
| 41663 | Fluorophore-1 | CCA GGA ACT ACT CTC TAC CTA AAC ACA | Quencher-1 | WT Probe | ||
| 41664 | Fluorophore-2 | AAT ACC CCA CCT GAC ACA GC | Quencher-2 | MUT Probe |
Reaction A
| Component | Final Concentration |
|---|---|
| Kapa Probe Fast QPCR | 1.00 X |
| ddH2O | |
| 41660 | 0.40 uM |
| 41661 | 0.40 uM |
| 41662 | 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.