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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
>chr15:45113088-45113234 147bp CCATCATTTTTGTGGCAGTG TCACACATAGGAAGCGCAGA
Mut= 154 bp
Wt= 147 bp
Fam=Mut
Hex=Wt
Sequence
Wt Sequence (deletion in lower case):
TGCTGTCCTGCCATTCCTATTGCTCCTTCATATATTTTTCTTTTATACACACAAATGTTACAGGTGGGAAATACATGTTCTTGTCACCAGAAAATATGTCACAAAAAATTAACTGATCTAAAGTTGCCTGCTAATGTATAAAACCTATTTGCATAAATTTCTAGTAAGAAAAAAGTGTCTTTTTAAAAGTCATAAGTAGGAAatctgtcagggaccaccatggacaaatatgaatatatattcctatattttgcacatatgaaagtgtgacctctctgtcaaatttaattctcttgtacgtctgtgttttttttttttttaattaatcacaaagttgtcttctttgaaagatacttcagaagaaaggagctgagtgaaactcttgactttaagaaggacacagatgaccaggaaagtcaacatgagagtgaacaggacttctcaaaaggaccttgtcccactgtccgacaaaagctctgggatattctggagaaacctggatcttccacagcagcccggatctttggagtaatctccatcatttttgtggcagtgtccatcgtcaacatggccctgatgtcagctgagcttagctggctcaacctacagctgctggagatcttggagtatgtgtgcatcagctggttcaccggggagttcgttctgcgcttcctatgtgtgaaagacaggtgccacttcctgaggaaggttccaaacatcatagacctccttgccatcttgcccttctatataactcttctggtggaaagcctgagcggcagccacaccacacaagagctggaaaatgtgggacgtctggtccaggtcttgaggctcctcagagctctgcgcatgctgaaactgggaaggcattctacaggtattcatatttcactcgtgccttttaatttcgcttttaaaataatctttagatatattggcacagaaccaccttacattcttactcaccctccaTCTATAAAGTACTAGGAAGAAAAAACTTCAAGATGCTATCATAAGCAAAGATATCTCCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCATATATATACACACATACTCTTTCTGTTCACAAGACAATTTGGACATAACAATTTGATG
This mutation is a 773 bp deletion beginning at Chromosome 15 position 45,112,795 bp and ending after 45,113,567 bp (GRCm38/mm10).
JAX Protocol
Protocol Primers
| Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
|---|---|---|---|---|---|---|
| 39053 | CCA TCA TTT TTG TGG CAG TG | Wild type Forward | A | |||
| 39054 | TCA CAC ATA GGA AGC GCA GA | Wild type Reverse | A | |||
| 39055 | CTG ATC TAA AGT TGC CTG CTA ATG | Mutant Forward | A | |||
| 39056 | GAG AGA GGA GAT ATC TTT GCT TAT G | Mutant Reverse | A | |||
| 39057 | Fluorophore-1 | CAT CGT CAA CAT GGC CCT | Quencher-1 | WT Probe | ||
| 39058 | Fluorophore-2 | AGG AAG AAA AAA CTT CAA GAT GCT AT | Quencher-2 | MUT Probe |
Reaction A
| Component | Final Concentration |
|---|---|
| Kapa Probe Fast QPCR | 1.00 X |
| ddH2O | |
| 39053 | 0.40 uM |
| 39054 | 0.40 uM |
| 39055 | 0.40 uM |
| 39056 | 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.