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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
>chr1:162722804-162722959 156bp TGCCTACTAACTCCAAGAATAGAGTC AAGCAGAATGCTGGGTTCC
Mut= 150 bp
Wt= 156 bp
Fam=Mut
Hex=Wt
Sequence
Wt Sequence (deletion in lower case):
TGTGTAACCCTAAAGGGCTTAGAACTTAACAGTGGATCAGGCCAGCCTCCAACATGAAGCTATTTTCCCACTTTCACCTTCCAAGTTGTGTAGTTTGAACTTTCTTCTCTTAACATTTTAAGTTTTTTACTTAAAATAGATTGCCTGAAAACATTTCAAAACTAGCATTTCCCCTTCCCATATTCccagtatatagtgttccatatttagaagttaaggggttctggtgggtgggtttgttttgttttttgagacaaattttgctggggtcccaagatggcttaatattttgagtggattgtttactttttaccttcaattaatgttgtaggttataaactgagtttatagagctctactttaacattggggactagtcagagattgttaactcctgttcatgtatattatttgttgaataaaatgtataatagagtgatgaacagtagatgttctaaggaaattgatgttactgatcttttagttgcagctcgacatgggttacaaagtcttggaaaagttggtatttcacggcgaatgcccccacctgctaaccttccaagtctcaaagcagagaacaaaggcaatgatcctaatgtgaatattgtgcccaaagatgggacaggatgggcatcgaaacaagagcaacatgaagaagaaaagtgagtcagaatctgttaagaataaaacacttctctcattgtaggaaactgccaccgctgtgcactgttcagccaatagaaacactctatgacacttataattcagagagttcttcctcagatgaaaacacaagtcagcttactctttgatgcctactaactccaagaatagagtcttggacttctcttaacactattacttagcaaaaacatgaaaaatgaacttaacattctaagaacaaatctacaagccaaccaatatttactgcaaactattatcTGAGTGTGGAACCCAGCATTCTGCTTTAATAAGTCCTTGATAAATGAAGAGTTAAAAAGATAATATAGATATTATAACTCTTTTGTGGTGTTATATTTAAAGTGTGAATAGACACATTTAGTGTAAATATATAATAACTGTGTATACACACTTTTAGTCGTTAGGCTCTTTGAGTTCAGTAGCATTAGCCATAAGCAC
This mutation is a 757 bp deletion beginning at Chromosome 1 position 162,722,830 bp and ending after 162,723,586 bp (GRCm38/mm10).
JAX Protocol
Protocol Primers
| Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
|---|---|---|---|---|---|---|
| 39080 | TGC CTA CTA ACT CCA AGA ATA GAG TC | Wild type Forward | A | |||
| 39081 | AAG CAG AAT GCT GGG TTC C | Common | A | |||
| 39082 | ATT TTC CCA CTT TCA CCT TCC | Mutant Forward | A | |||
| 39083 | Fluorophore-1 | AGC CAA CCA ATA TTT ACT GCA AAC | Quencher-1 | WT Probe | ||
| 39084 | Fluorophore-2 | TTC CCC TTC CCA TAT TCT GAG | Quencher-2 | MUT Probe |
Reaction A
| Component | Final Concentration |
|---|---|
| Kapa Probe Fast QPCR | 1.00 X |
| ddH2O | |
| 39080 | 0.40 uM |
| 39081 | 0.40 uM |
| 39082 | 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.