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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
>chr3:116948069-116948166 98bp GCCAAGTCAGTGGGAAAAAC ATTTGCCAGCAATTCCCAAG
Mut= 106 bp
Wt= 98 bp
Fam=Mut
Hex=Wt
Sequence
Wt Sequence (deletions in lower case, insertion with carrots ^g^ (AT insertion):
CCACCTTATTTTGACAGATTTTTACATGATCATAATAGTTATTTGTAATTATTAAAATATTGATTTGGTTTCTTTTTAAAGAAGAAAGGGGTCAACAAGTTTACAAGGAAGGGAAAATGCCAGATTTATTGAGCCAAGTCAGTGGGAAAAACTAATCTCTGTTGATATG^cccgctgtcgccatgcataagacttgaaagcaataggagatcttgggaattgctggcaaatataagaacatacgtttatattccctgacatcagaaaaaagccctgagggagaaatggctcctggatggaatcggcagtggaaaagaacacgaagagatgaagaagcaaaatcagcaagaccagcaccagacccaggttctagagcaaagcatcctcaggtacggccctcactggggtacttctcaaaccgagctccttccctaaa^AGGGAGCTGGGgtGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGCACACGCATGCTCTCTCAGGTATACTCACACATGTAGAAGCCAGATATGAATGCTGGCATGTCTTCCTCAGTTGCTTCTCCCAGATTTTTAATATTTTTATTAATTCTTGAGAACTCCA
This mutation is a 266 bp deletion beginning at Chromosome 3 position 116,947,864 bp and ending after 116,948,129 bp (GRCm38/mm10).In addition, there is an AT insertion at the deletion site as well as a 2 bp deletion GT 11 bp after the 266 bp deletion that will not alter the results of the deletion.
JAX Protocol
Protocol Primers
| Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
|---|---|---|---|---|---|---|
| 40546 | GCC AAG TCA GTG GGA AAA AC | Common | A | |||
| 40547 | ATT TGC CAG CAA TTC CCA AG | Wild type Reverse | A | |||
| 40548 | CTG AGA GAG CAT GCG TGT G | Mutant Reverse | A | |||
| 40549 | Fluorophore-1 | CTG TCG CCA TGC ATA AGA CTT | Quencher-1 | WT Probe | ||
| 42053 | Fluorophore-2 | ATA TGA TAG GGA GCT GGG GTG T | Quencher-2 | MUT Probe |
Reaction A
| Component | Final Concentration |
|---|---|
| Kapa Probe Fast QPCR | 1.00 X |
| ddH2O | |
| 40546 | 0.40 uM |
| 40547 | 0.40 uM |
| 40548 | 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.