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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
Sequence
Wt Sequence (deletion in lower case):
TAAATACAAATACAAATATTCCATGCTCTGTAATAAATATTTGTGTTATTTAATTATTGAATTGAGAAGCTTTTAGCATCACTTTCATCCCTAAAGTACTTGAAAGTCTGACCTCCTTTTATAGATATTGTCAGACTTTAATGTTAGGACATCATTTTAAAGACCTTCACTTAATGTCTTAGACCCCACTTGGACagtaagcagttccttgagtttattataaattttggcaggtgtggaaaattctaagtcaccaggagaatcttgattcagttactctccaagggcactaattggccctttctacagtgctcagggaattccaaggagacactgaccgcctggcatagttttgcccattgagatgcagcttagctgtctcttcttgtttttgttttgcaagacacttgttttaacttcttctacttttgtgttaattgtagtttctgatgaagatttgaaaagaagagtagctgaggagctggcattggagcaagccaaaaaggagtcagaacaccagagacggtgagccccatgggacCTGAGAAGACTTGCCTGGAGTTGTGATATGTAGCATGCTTAAGGAAGACTTATTTATTTTATTTTATGTGGATGAGTGCCGTGCTTGCGTGTATGTCTGTGAACTTTCTGTGTTCCTGGTACCTGTAGAGGTCACAACAGGGCATTGGATTCTTTGGAACTGAAAGTAGAGACAGTGTGAGTCACCATGTGGGTCCTCTGCAAGAACAGCAAATGCTCTTTTAACTGGTGATTTAAAAC
This mutation is a 346 bp deletion beginning at Chromosome 6 position 33,008,467 bp and ending after 33,008,812 bp (GRCm38/mm10).
JAX Protocol
Protocol Primers
| Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
|---|---|---|---|---|---|---|
| 67868 | Fluorophore-1 | CCA CTT GGA CCT GAG AAG ACT TGC | Quencher-1 | MUT Probe | ||
| 68834 | AGC ATG CTA CAT ATC ACA ACT | Mutant Reverse | A | |||
| 68835 | TTT AAA GAC CTT CAC TTA ATG TC | Mutant Forward | A | |||
| 69773 | CTT TCT ACA GTG CTC AGG G | Wild type Forward | A | |||
| 69774 | CTG CAT CTC AAT GGG CAA | Wild type Reverse | A | |||
| 69775 | Fluorophore-2 | AAT TCC AAG GAG ACA CTG AC | Quencher-2 | WT Probe |
Reaction A
| Component | Final Concentration |
|---|---|
| Kapa Probe Fast QPCR | 1.00 X |
| ddH2O | |
| 68834 | 0.40 uM |
| 68835 | 0.40 uM |
| 69773 | 0.40 uM |
| 69774 | 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.