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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
>chr6:35186591+35186725 135bp GCTTCTCCTTGCTGGTAAGTTG GCAATGCGAACTCAGGTATG
Mut= 141 bp
Wt= 135 bp
Fam=Mut
Hex=Wt
HOM Lethal (DJL 7-29-19)
Sequence
Wt Sequence (deletion in lower case):
AAGGAGGTTTTAAGGGGCCTAATGAGGTGGCTTAGTACTAGAACACTTGCCTAGTGTGTGTCCATTTGTGGGTTACATCCCTACCACCACACATATAAAAAGAGTTTTGTAGGATATAAAATCATGTTACTAATTTATGATTCACTCTACaaagtaatactattgcttgttacttaatatagttcatatgtccatttgaattattgtatgtaactagaacattcacttagatatgtggaaatcagaatagtatttgtatttttcctggtttttatggagcttagcagatagttttgagaaggtagtactgggaaagatgaaggtggtgccacgtgttgcctcatatcccctagttctggataactagataagaaacacttcagtttgtcatgctaccaggcttcattgtttttgttcgcatgcatgctggccatgactctacttgcctggcctccaaatataggattaaaggcatgtgctgccttggcaaacgaggctgcgttgtcatgaactgacttctgctaggagcactattttcattacttaattataattttatcattctagcccaaaaatgttcaacagcatgagaaaattcagaaagccagttcagagggtgttgcgatccaggggcaacaaggaaccagacttcttcctgagcagctcattaaagaagcatttatcatcagtgacctttttgatattggggaattgtcagctgttgagcttctccttgctggtaagttgacatttagctggaagtgtgatatgtagtcagaatcatgaagctagaacccctaTCTTGGACTTTGGTTTTCTTTTAAAGATTTATTTATTTCGCATACCTGAGTTCGCATTGCTGTCCTCAGACACACCAGAAGAGGGCACTGGATCCCATTAGAGATGGTTGTGAGCTGGGATTTGAACTCAGGACCTCTGGAAGAACAGTTAGTGCTCTTAACCACTGAGCCATCTC
This mutation is a 605 bp deletion beginning at Chromosome 6 position 35,186,016 bp and ending after 35,186,665 bp (GRCm38/mm10).
JAX Protocol
Protocol Primers
| Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
|---|---|---|---|---|---|---|
| 38394 | GGG TTA CAT CCC TAC CAC CA | Mutant Forward | A | |||
| 43456 | GCT TCT CCT TGC TGG TAA GTT G | Wild type Forward | A | |||
| 43457 | GCA ATG CGA ACT CAG GTA TG | Common | A | |||
| 43458 | Fluorophore-1 | AGC TGG AAG TGT GAT ATG TAG TCA GA | Quencher-1 | WT Probe | ||
| 43459 | Fluorophore-2 | ATG ATT CAC TCT ACT CTT GGA CTT TG | Quencher-2 | MUT Probe |
Reaction A
| Component | Final Concentration |
|---|---|
| Kapa Probe Fast QPCR | 1.00 X |
| ddH2O | |
| 38394 | 0.40 uM |
| 43456 | 0.40 uM |
| 43457 | 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.