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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
>chr1:36357723-36357823 101bp TTTCCCTCTATAGCCTTGTAGTTC CAGAAGGTACCCTTGGTCCTC
Mut= 100 bp
Wt= 101 bp
Fam=Mut
Hex=Wt
Sequence
Wt Sequence (deletion in lower case):
TGTGTTGTAATCCCAGCACTATGGAGGCCGAGGCAGGAGAAGAGTTTAGAGTTTGAGTACAGTTTGAAGTGAGACCGTACTGGAAATAAATAGAACTGAAACTTGTCTTTTGTTACAAATGTGATTATTTTAGCCACCAGATAAATAAATAGCTTTTGAGCACTGAAAAAAAAGCTCCATGAtttgttgtgacttgcggtgtatggtaaggtgattcattggcggaattgggaccaaaagctagagttcatgctttagcttagtggccttagactttttgtaatgagaacaaaaatatgacagtatctctttatatggtggacatacatgggaaatgtcctcagggttgcattttaattcctcagagtttctgttataggactggctggacagtggctcagaataagctatttaacaagatcctcaaagccctgcagtctgaccggcttgcacgattggccaatgaaggggtaagacattggaagtctgtctatagttgactctcttagttttggcctcgggggttgttgtgtcctgtgtgctctctcccctcagcccccagcttgctcacggagcgtaactcttgttttattctttttgtgaacacctttcaggcttgcaatgagcctgtgctgcgtcgcgtggctgtggacaagtgtgcaaggagggtgcggcaggcgctggcaagtgtcagctgggacaccaagctgacccagtggctgcacaccaccctcgtggagaccctgagcctgcccatgctggctgcttacttggatgccctgcagacactgaaagggaaggtaagactgtgcttggtttgcccgagtggcagcagccagggtttgctgtgagttcagagggtttgcacaaggccctgtgactaggtcagcacagaagctaacaatttggctattataatggaaaaggggatcattttcccttctatttggtataaatttcttcagtcgtgccatttttttggataaagtgtggtatctgaaaaccaggatacataatttccctctatagccttgtagttcttaccttgagtcatcgctgacttgaactgtggtggaagagACGAGGCTGAGCAGAGGAGGACCAAGGGTACCTTCTGAGTTAACTCTCCTCATGAAGAGATGGACTGTAGGCTGAGTATGGTGCTACACACCTTTAACCACAGAACTGTACAGGCAGAGGCAGGTGGCTCTCCATCACTTGGAGCTTGGTCTACACTGCAAGT
This mutation is an 898 bp deletion beginning at Chromosome 1 position 36,357,760 bp and ending after 36,358,657 bp (GRCm38/mm10).
JAX Protocol
Protocol Primers
| Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
|---|---|---|---|---|---|---|
| 43995 | TTT CCC TCT ATA GCC TTG TAG TTC | Wild type Forward | A | |||
| 43996 | CAG AAG GTA CCC TTG GTC CTC | Common | A | |||
| 43997 | TGT GAT TAT TTT AGC CAC CAG A | Mutant Forward | A | |||
| 43998 | Fluorophore-1 | CTT GAG TCA TCG CTG ACT TGA A | Quencher-1 | WT Probe | ||
| 43999 | Fluorophore-2 | AAA GCT CCA TGA ACG AGG C | Quencher-2 | MUT Probe |
Reaction A
| Component | Final Concentration |
|---|---|
| Kapa Probe Fast QPCR | 1.00 X |
| ddH2O | |
| 43995 | 0.40 uM |
| 43996 | 0.40 uM |
| 43997 | 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.