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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
>chr16:11162786-11162895 110bp CGGAAACGCACAGTTTAAGA CCTCCACTTCCCAAGTTCTG
Mut= 101 bp
Wt= 110bp
Fam=Mut
Hex=Wt
Sequence
Wt Sequence (deletion in lower case):
TTGAACTCAGGACCTTTAGAAGAGCAGTCAGTGCTCTTACCCACTGAGCCATCTTGCCAGCCCGAGTGTTGGATCTTTGAGGTGCCATGCGGCTTCTGGGAACTAAGTTCAGAACACCAAGCATTCCTAACTACTCAGCCATCTCTCCAGCCCCGGAAACGCACAGTTTAAGATAACTTCTGGCtgaattatgctgatagagggcctgtcatggtacagcatggtagagcgttcttgtaatctcagaacttgggaagtggaggcaagctgatctcagtaagttccaggccaatcagggttgctgccttagaactaacaaaacttttagtagaacttggtaattacaggagatttgcttcttcctctcaggcctccgctgtcatatcctgggacccaggagcagtatgcggtaaggatgtggggagccctatggctgggcacagggcctcaagagctgtaatctgcagcaggatttggcagtctagccttgactctcacacctgaccagctgccctccttcataaagagagctttactggaacaccgcggccacaggcggttgttcatgggttctctgtggtgcctgtgtgagacagggcagaactcaaatgtcccacagtgctttaaatattctctttggcatttacagaaaaagcttgccaatctcttctatatagtgtaccctttcggggcatttctggacAGTTGTCCTGCATTTTCTTTCCTGTGTTAGTGTAGTTTGAATTAGTATGCATTTTTAGTCCTTTGCCAGTTAACTAACATTTTATCAAAAATAGGTGGGATAGAATTCAGCAGAGAATGTGGGGTATCATTATTTTTGTTTTTTAAGTCCTGAAAGCAGTGAAGGAGGATTGTCTACATAGGGAG
This mutatuion is a 519 bp deletion beginning at Chromosome 16 position 11,162,346 bp and ending after 11,162,864 bp (GRCm38/mm10).
JAX Protocol
Protocol Primers
| Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
|---|---|---|---|---|---|---|
| 41667 | CGG AAA CGC ACA GTT TAA GA | Common | A | |||
| 41668 | CCT CCA CTT CCC AAG TTC TG | Wild type Reverse | A | |||
| 41669 | ACT GGC AAA GGA CTA AAA ATG C | Mutant Reverse | A | |||
| 41670 | Fluorophore-1 | TGA TAG AGG GCC TGT CAT GG | Quencher-1 | WT Probe | ||
| 41671 | Fluorophore-2 | CTT CTG GCA GTT GTC CTG C | Quencher-2 | MUT Probe |
Reaction A
| Component | Final Concentration |
|---|---|
| Kapa Probe Fast QPCR | 1.00 X |
| ddH2O | |
| 41667 | 0.40 uM |
| 41668 | 0.40 uM |
| 41669 | 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.