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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
>chr18:63030212-63030311 100bp CCACGGTCCAGCTTCTCTTC GAGGCACCAGATCAATGAGA
Mut= 99 bp
Wt= 100 bp
Fam=Mut
Hex=Wt
Sequence
Wt Sequence (deletion in lower case and insertion with carrots ^g^ (AGAATGGGGACGAGGAGTGTTAGGATATTAATAAAGAACACTATTAGGGAGAGGATTTGAACCTCTGGGAACAAGGTTTTAAGTCTTACGCAATTTCCTGGCTCTGCCACCCTAATAACCTTCTCT insertion):
GGAGCTAAGGTTGGGGTCACCTGTGCATGGTAAGTGGGTCAAGGCTAGTGCTAGTGCTAGCAGTGAGCACTCAGATGTGTTCTAGAAGGTTCTAACAAATTGAGAGGTCATTGTCTCATCCTTACTCTATCTGCCTCACTCTACCTGCCTTGGCTTAGTTACTCATACAAACTCTTTTCACCCGCAAACGTCTCCACTCT^cctaggacactcacaattctgtacaagcttccttagacataaatcccttggctttccaacgtcaagggattgaatgttggaagggacactgtaagaaagacagccagttggcccacccaagatgctagctagcctcagctgtcatcttctcttcttctaacagtgtcatggtctatgggacgaggatgacattgttgacagtaacacagacaaggaaggatcggatgacgagctgtccctcgatcagggcagaagaggctcctcagactcactgaagtctatcaacctggctgcctctgtagagtctgtccacgtgaccttccccgagcagccagcggccatcaggagaaaacgctcctgcagtagctctcagatctccccacggtccagcttctcttcaaataggtccaaaagaggtatctgttgcatggccattaggttga^AATGAGCCCCACATAGTCTCATTGATCTGGTGCCTCTGCATCTGGGGAGGGAAGTGGTTCGGTGCTATCAAAAGACGATGTTGTGACATGGAAGAAACATGCTCTGGCATATGACAGACCCAAGTTCAGAACCTGTTTCATC
This mutation is a 443 bp deletion beginning at Chromosome 18 position 63,030,248 bp and ending after 63,030,690 bp (GRCm38/mm10). In addition, at the deletion site there is a 126 bp insertion (AGAATGGGGACGAGGAGTGTTAGGATATTAATAAAGAACACTATTAGGGAGAGGATTTGAACCTCTGGGAACAAGGTTTTAAGTCTTACGCAATTTCCTGGCTCTGCCACCCTAATAACCTTCTCT) that is from the mouse mitochondrial genome (ChrM 2666-2791).
JAX Protocol
Protocol Primers
| Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
|---|---|---|---|---|---|---|
| 42139 | CCA CGG TCC AGC TTC TCT TC | Wild type Forward | A | |||
| 42140 | GAG GCA CCA GAT CAA TGA GA | Common | A | |||
| 42142 | Fluorophore-1 | CTG TTG CAT GGC CAT TAG GT | Quencher-1 | WT Probe | ||
| 43199 | GGT TCA AAT CCT CTC CCT AAT AGT | Mutant Forward | A | |||
| 43200 | Fluorophore-2 | CCT CGT CCC CAT TCT AAT GA | Quencher-2 | MUT Probe |
Reaction A
| Component | Final Concentration |
|---|---|
| Kapa Probe Fast QPCR | 1.00 X |
| ddH2O | |
| 42139 | 0.40 uM |
| 42140 | 0.40 uM |
| 43199 | 0.40 uM |
| Wt Probe | 0.15 uM |
| Mutant Probe | 0.15 uM |
| DNA |
Cycling
| Step | Temp °C | Time | Note |
|---|---|---|---|
| 1 | 94.0 | -- | |
| 2 | 94.0 | -- | |
| 3 | 65.0 | -- | -0.5 C per cycle decrease |
| 4 | 68.0 | -- | |
| 5 | -- | repeat steps 2-4 for 10 cycles (Touchdown) | |
| 6 | 94.0 | -- | |
| 7 | 60.0 | -- | |
| 8 | 72.0 | -- | |
| 9 | -- | repeat steps 6-8 for 28 cycles | |
| 10 | 72.0 | -- | |
| 11 | 10.0 | -- | hold |
JAX uses a very high speed Taq (~1000 bp/sec), use cycling times recommended for your reagents.
JAX uses a 'touchdown' cycling protocol and therefore has not calculated the optimal annealing temperature for each set of primers.
Strains Using This Protocol
This is the only strain that uses this protocol.