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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
>chr1:192123965-192124097 133bp TGCTGAGCCGAGACTTTTCT ATGTCTGAGTGTGGAGGACTAGG
Mut= 125 bp
Wt= 133 bp
Fam=Mut
Hex=Wt
Sequence
Wt Sequence (deletion in lower case; and insertion with carrots ^g^ (C insertion):
TAAGAACTAGATGTAAAACTAAAGTGATTTTAAATGGTCACTGTTAAGTACTGAGCAGCTCTTGGCTGCCTGAAAGAGGCCAGACTCAAAGGACTATATGGTGTGATTCTCTAGTGTGAAGTTGATGTTGATTTCATTTAGACACCAAAGGCTCTAAACTAGAGAAAAGAGAACTGAGTTACTCTTAAGAAACACATGCTGAACGCTAGCAGAGCCATGCTGGGACCGTGGAGGACATTCTCATTAAGTTAGTTTGTGACATCTTCCCTCTCCCCCAGAACCATGGGTTCAGCAGTGTCAGTGACTTTGGTATAATGGAGTTCTATGACTATTAGCAGTACTTTTTAGTTGTTAGTAAAGGTACCACG^acatggctttcatttatgccttgattatcaaagaatatatagttaatcatttggtgatcagagttgatgaactaaactttccaacagtaacaggagttttttttaaaagcttccagataagacaattgcaagtcttgtaaagtattactattcttggaaaaaaactcgctcaaggacaagtttgatggatcgccaggctcgtaaactagccaatagacacaatcagggtgacaggtatgtcatacgtttggcctctgttgcactgttggggacttttcacgggactgtatagttttcataagacagaaagcaactgcagcactgtgtctgctgtggagcgcgcagagttgtccgaagctctgactgtaggagctggacatgctgagccgagacttttcttcttccagcgttgcttgttcagtgtgactgaaagacaaactgggacgtgattccatgctctagcaccagccccgt^GAGGCCTGGTTCCATCCTAGTCCTCCACACTCAGACATATTAAACAATATATCCTGTGCAAGATGGCCTTCTGCATTCCCTGAAGTAACAAAGTCCCCCCTACCTTAATGTGCTTTGTCCGTCTTTGCACCAAGCACAGAACTTAAGTCACCAGCGTTACCACAGATAGCGAGTGCCCTCTCCCACGCGGTACTTCAAAGGTTTTTTTGTTTTGTTTTCCCCACAAATTCTCTTACTTTTTAGCTAGAAATCTTTATTAAATGATTCTGTAAGCCCCAAAAGACGGATGTTTCTTAGATCCCTATTACAACTGTTCTGCATAGTGACAGCTCTGAGGAAGATGTGTTAGAGTTACGAGAAGACATGCTGATCTTGTCTCTTTCAGTGATGATGACGTAGAA
This mutation is a 474 bp deletion beginning at Chromosome 1 position 192,124,003 bp and ending after 192,124,476 bp (GRCm38/mm10). In addition, there is a 1 bp insertion C at the deletion site.
JAX Protocol
Protocol Primers
| Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
|---|---|---|---|---|---|---|
| 48453 | TGC TGA GCC GAG ACT TTT CT | Wild type Forward | A | |||
| 48454 | ATG TCT GAG TGT GGA GGA CTA GG | Common | A | |||
| 48455 | ATG GGT TCA GCA GTG TCA GT | Mutant Forward | A | |||
| 48456 | Fluorophore-1 | AAC TGG GAC GTG ATT CCA TG | Quencher-1 | WT Probe | ||
| 48457 | Fluorophore-2 | TGT TAG TAA AGG TAC CAC GCG AG | Quencher-2 | MUT Probe |
Reaction A
| Component | Final Concentration |
|---|---|
| Kapa Probe Fast QPCR | 1.00 X |
| ddH2O | |
| 48453 | 0.40 uM |
| 48454 | 0.40 uM |
| 48455 | 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.