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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
Mutant= 100 bp
Wild Type = 87 bp
>chr17:36872134+36872220 87bp CTGAGGAGCAGGCAGGAGTA AAATTCCCACTTCCCTCTTGA
Sequence
Wt Sequence (deletions in lower case):
CTGAGGAGCAGGCAGGAGTAGGGGGCTCTGATTTTACTgcctaaccttgggaggtgtgggcatagatcaagagggaagtgggaatttataaggaaagggaagaaaggacagatgagatgccacaggaagagggggaaaagatgggagaagatctcgctgtgaggagagtaaaatgttctccctggctctcatcttcccttctcagactcaggtggccaccaagaggcaacaggtgatctcacagtttgcgcatctgagccagttcctgcagcaacagcagactgccctcttagcacaactggaggggttagacggggacatcttgaagcagcaggaagagtttgattccctggccactggggagatctgccggttcagcaccctgattgaggagctggaggagaagaacaagaggacagccagggggctcctgacggtgaggcctggcctccagggtctctggaattactgcctaattggtacctccttgacatacatgtttaggcactgaagatggaggaggtacatggggaaagggggttggattgtaatctgtgcttccccaagagctgcctggtcactgtgctctctgttgttggtctctccttcctgccctccgtagaatggagaggaaatgaggatatgacagtaaaaggaggtggaagttgctcctgagacgtatcacacactgtgcaaacaagtgccaggtgttgcctccaaagcaaaactttcccctccatggccaggaagttcttcctagactgtaccacttccttgaactggaggagagttattggggttccaggagcatgctttagggagatgcactgggtactgggaaataagagactttggtgtagaacttgctcacttttgcctatagctggggagaagtaggtcgtatcatcaactctgtccatgaaacaaaggtctgggttctggtcttaatgtccaaggctgggaatacggcagtattttgggttttatggtcataacaaaagtgggagagaaaaactagaacctaattctctctctctctctctctctctctctctctctctctctctctctctctctctctgtccatctgttcctccctctctccctccctcctttcctttcttcctttcccctctcaggatatcagaagcactctaataaggttagtgataaagtatctacttttttcttccccatagacattcatccatacaccacgtacctagttattgacgtgaaaataaaccactttctcagtttagggtacagtcatatataaatgtacaaatgctcaaatgtgttctcagatagtgctgtagatctacccacttgtgcagcagtggggtccatctgagcggatccaagtctctatgtgggaagttgctcctcaagGCCTTGTTAGAAAAAGATGGCTGTCTGGGGACATCATTAGAAGACAATAATTCTGGGGCTGG
This mutation is a 1354 bp deletion beginning at Chromosome 17 position 36,872,172 bp and ending after 36,873,525 bp (GRCm38/mm10).
JAX Protocol
Protocol Primers
| Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
|---|---|---|---|---|---|---|
| 65358 | CTG AGG AGC AGG CAG GAG TA | Common | A | |||
| 65359 | AAA TTC CCA CTT CCC TCT TGA | Wild type Reverse | A | |||
| 65360 | CCA GCC CCA GAA TTA TTG TC | Mutant Reverse | A | |||
| 65361 | Fluorophore-1 | CTA ACC TTG GGA GGT GTG G | Quencher-1 | WT Probe | ||
| 65362 | Fluorophore-2 | CTC TGA TTT TAC TGC CTT GTT AGA AA | Quencher-2 | MUT Probe |
Reaction A
| Component | Final Concentration |
|---|---|
| Kapa Probe Fast QPCR | 1.00 X |
| ddH2O | |
| 65358 | 0.40 uM |
| 65359 | 0.40 uM |
| 65360 | 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.