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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
>chr1:58493780-58493931 152bp AAAAACATTCTGGGAAGACATC TGTGATCAACAGAGAGCAAAGT
Mut= 147 bp
Wt= 152 bp
Fam=Mut
Hex=Wt
Sequence
Wt Sequence (deletion in lower case):
GCAGAAGGATTTCTGAGTTTGAGGCCAGCCTGGTCTACAGAGTGAGTTCCAGGTCAGCCAGGGCTACACAGAGAAATCCTGTCTGGAAAAAAACAAAAAAAACAAACAAAAAAGATAAAAACATTCTGGGAAGACATCTTAACGGCTTTTTTTAAAAGTTGTACTCTTAATGTAggtaggtttatatacctttctgataaaagtagtacaggtgtttgttttaaggagttcatataagtgaaagctactttgctctctgttgatcacaaaagtgcttttatttttccccgattacagctttagaactagcaaaaacatcaggaaaaaaggatccacttgactcaaatgatcctgaaatcacaaaaaacattgctcaaaaaagtaaaggtaagataccatcaggtgatctgtgtgtgctacttttttttccttaaatctaagtgaatttccatgtattttttttgttttcagaaaatgtatttaacagaatgcaatttttgttttgttttgttttgttttgggtttttttggtgttttcaagacagggtttctctgtatagccctggctgtcctggaactcactctgtagaccaggctggcctcaaactcagaaatccgcctgcctctgcctcccgagtgctgggattaaaggcgttgccatcactgcctggccagaatgcaatttttaatatatctatacatatcagtcaataaagggcagaaagtcatctgtgccaccctatgttgggagaattgacgaggaaacctaaaacaatcatcaagtcctataatcatttgtctgagttagccagtttctcatgcaaggtttggaagttgaatggcttgttgcaaattgcagagcatctatttttgaattacccattccagataagagagacagtgtgctttctggtccaaatgaaaaaggctcggggttttgcttccctgttagaatgccacagaaattcaagaatgctgaaaatgttgccactggttagagtagccatataattcttggtgaccatttagcctgtagttaaatggaaactcagtacaatacagtggcttgagtgtaaaacgtgcagctttgctggcaacaaaactatgtcatgatctaagatgaaatatttgtagtaaatgtgacctttgttccgtttagggcattctacttcagagaaagctccactggtggtaagtaactgacaaaacaagcaagtgtgataaatggtagctttctgtcttcgtttcagtgttaaagtttgtgccagttatatacctgttatctgaggttcataaaggacagatgacttacagagtacctcacttggaataaaactggagaatgaaccctggtttcccCTACATagctgttctctgctcttcctgctccttcacagctatcctgcgtatttgtaacttagtagacctgacctcggaatTTGATGAGTTTGGTTTTGTATTTATAATGTTCTCTTCTTAGAGTATTTCGTCCATTAAAGTCTATATTGGCTTGGGTAGGTTCATTCCAGTTTAGCCAAAAGTAATCCACAGTTTTTGTCTCCTTTGTGTTGTAGTGATTTTG
This mutation is a 1259 bp deletion beginning at Chromosome 1 position 58,492,609 bp. There is a 6 bp endogenous retention (CTACAT) after 58,493,784 followed by 74 bp of the deletion and ending after 58,493,873 bp (GRCm38/mm10).
JAX Protocol
Protocol Primers
| Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
|---|---|---|---|---|---|---|
| 46346 | AAA AAC ATT CTG GGA AGA CAT C | Common | A | |||
| 46348 | TGT GAT CAA CAG AGA GCA AAG T | Wild type Reverse | A | |||
| 46349 | GAA CCT ACC CAA GCC AAT ATA GA | Mutant Reverse | A | |||
| 46351 | Fluorophore-1 | AGT AGT ACA GGT GTT TGT TTT AAG GAG TTC | Quencher-1 | WT Probe | ||
| 46352 | Fluorophore-2 | CTC TTA ATG TAC TAC ATT TGA TGA GTT TGG | Quencher-2 | MUT Probe |
Reaction A
| Component | Final Concentration |
|---|---|
| Kapa Probe Fast QPCR | 1.00 X |
| ddH2O | |
| 46346 | 0.40 uM |
| 46348 | 0.40 uM |
| 46349 | 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.