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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
Mutant= 130 bp
Wild Type = 133 bp
>chr1:181600444+181600576 133bp AATTCTCATGGTGCCGAGTG TGCTGAGTAGATGCCTGTTCTG
Sequence
Wt Sequence (deleted region in lower case)
AATGAAATCACGGGTCTGGCGAGGTGTCCCAGCAGTTAGAACATGTACTGCTCTTGCTGAGGATCCCAGCAGCTGCATTGGGTGACTGACAGCCCCATGGAACTCCAGCTTGAGGGAATATCTGCTACCTCTGGTCTCCTTGGGCACCTGCACTGACGTGCGTATGTGCACACACACAATTTAGTAAAATAATTCTTTAAAACTACTAAAAAGTGCATTGGCCTCCAAGTCCTCTTTAATTCTCATGGTGCCGAGTGCTGACGGCCTCGGCCTCctctctataattgggtcagatatagttgtttaggagctgtttttgtaatagagacatcttgatggaaaggaagtcagaacaggcatctactcagcatattaacagattataaaggtttcatggttgaaagtcttcagtgtaaggttgccatcactgaccactataggcaggtgatctaggaggtctcacgttgacttggacttgaatgacgtgattctgttttttaacgttaggaagaccatcactatcatctgaagttggagtcataatctgtgatatcagtaatccagcctcacttgatgaaatggctaaacaggcaaagcttgtcctcaactgcgtaggaccggtaagtagtcggcctttctttgtatcagaacaaacagtccattcattcctaacaaaatctggtattttacagtccagagggacacacgggtggagggcagatggcagattgaaggccttccctccacagggaagtctttccattgatatgatagtcttccaaagtagtgacaggtctttgagaccctattcagagaatgtgctgattggcaccccttaccctgtgtccccCTCTCTAATGTAAAAATAAAATAAAAGTTTGATGAGCTAGAAAGATGGTTCAGTGGGTAAAGGCATTTGCCCTGCAACCCGACAAGTTTTGTTCAGTCTCTCAGACCCATACAGTGGAAGGGGAGAACTGGTCCCCAAAAGGTGTCCTGTGATCTGTACCCTTGCACCTTAGCATGGGTACATGCATGCATGTGTGTGTGTACACTCCCATACACAGTAAATACATGTCATTTGACATTTTTAAACGTTTGCATTTACAGTCCCAGCTTCTTTGCTTTACTCTTGTATCATTATGTAGAT
576 bp deletion beginning at Chromosome 1 positive strand position 179,670,350 bp and ending after 179,670,925 bp (GRCm38/mm10).
JAX Protocol
Protocol Primers
| Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
|---|---|---|---|---|---|---|
| 38336 | AAT TCT CAT GGT GCC GAG TG | Common | A | |||
| 38337 | AAC AAA ACT TGT CGG GTT GC | Mutant Reverse | A | |||
| 38338 | TGC TGA GTA GAT GCC TGT TCT G | Wild type Reverse | A | |||
| 38339 | Fluorophore-1 | TGG TTC AGT GGG TAA AGG CA | Quencher-1 | MUT Probe | ||
| 38340 | Fluorophore-2 | AGA GAC ATC TTG ATG GAA AGG AAG T | Quencher-2 | WT Probe |
Reaction A
| Component | Final Concentration |
|---|---|
| Kapa Probe Fast QPCR | 1.00 X |
| ddH2O | |
| 38336 | 0.40 uM |
| 38337 | 0.40 uM |
| 38338 | 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.