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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
>chr4:155731509+155731619 111bp CACTAGGCTGGTAGACTTATGCTG GCTTGCTTCTCACAGGTTGTC
Mut= 98 bp
Wt= 111 bp
Fam=Mut
Hex=Wt
Sequence
Wt Sequence (deletion in lower case):
GACCATCCGTCGTCTAAGGTCTACTCTGTGCTGCTTTGGACACTTAGAAACAAGCATCTGTGTGGCCTGATCTGTCTTGAGAGGAGACAGTCCCTGCCATGCCTCTACTTTCCAGTTACAATGGCAAGGCTTTCTGGAGCCATTCTGTGGGTTTGGGTTATCGTGGGAGTTATGATCTctctacgaagtcagcaccttccctgtgcttcccatgttagggcagtggtgaccctgcttgtgttcaggcctagagctatgttgcccctgttgacaatggccaacttggaaccccctagaggcaaggggccattgacaggtctaggttagtaactatctttaatccaaagctccttgactctggtgttcagctatacacaaaatggaattttacatatgttggacagaaataaaaggatcaagccccgtccagaaaggttccagaactgcacggacctgtttgacttgatcctcacttgtgaagagagagtctatgaccaggttgtagaaggtgagtgcacagagcctgacctgctgttcaggttgtaccaccaacactaggctggtagacttatgctgccctgggcgacagtgatggagacagagggtgcccccacatcCTCTGACCTGTATGGACATCTGGAGGACAACCTGTGAGAAGCAAGCAGAAGACTGATGTCCAGCAAAACTCAAGCCTTGTGGCCTTCAATGTTTGCTTTTCCTATGTTAGGCCTGACATTTTATAGAACATGCTATTGGTGCCAATACTGACTGGCATAGGATGGCTTGAGGATTCTATCTAGGATTCTTA
This mutation is a 439 bp deletion beginning at Chromosome 4 position 155,731,135 bp and ending after 155,731,573 bp (GRCm38/mm10).
JAX Protocol
Protocol Primers
| Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
|---|---|---|---|---|---|---|
| 44545 | CAC TAG GCT GGT AGA CTT ATG CTG | Wild type Forward | A | |||
| 44546 | GCT TGC TTC TCA CAG GTT GTC | Common | A | |||
| 44547 | AGG CTT TCT GGA GCC ATT CT | Mutant Forward | A | |||
| 44548 | Fluorophore-1 | CGA CAG TGA TGG AGA CAG AGG | Quencher-1 | WT Probe | ||
| 44549 | Fluorophore-2 | CGT GGG AGT TAT GAT CTC TCT GA | Quencher-2 | MUT Probe |
Reaction A
| Component | Final Concentration |
|---|---|
| Kapa Probe Fast QPCR | 1.00 X |
| ddH2O | |
| 44545 | 0.40 uM |
| 44546 | 0.40 uM |
| 44547 | 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.