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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
>chr16:20654328+20654457 130bp GGCAGAAGCATCTTTCTTTGA AAAGTTCCATGCTAAGCCTCA
Mut= 133 bp
Wt= 130 bp
Fam=Mut
Hex=Wt
Sequence
Wt Sequence (deletion in lower case):
TTTTTTTTTTTTTTTTTTTTTTGGTTTTTCGAGACAGAGTTTCTCTGTATAACTCTGGCTGTCCTAGAACTCATCTGTAGACCAGGCTGGCCTCGGACTCAGAAATCCACCTGCCTCTGCCTCCCGAGTGCTGGGATTAAAGGCGTGCGCCACCACTGCCCGGCAGAAGCATCTTTCTTTGATTCCCCcattgtttattactcttgagttaggttagcttcagagggagctagcaacaccaactttagggtttttttttttttttttttttgaggcttagcatggaacttttctgtatagagactagtatatattgaattttataggtgaattttgtggacagtaatcttgattctgtaatggcacatgctgttttcctagtgttttgcagctgacatcatctctgttttggccatgaccatgagtggtgagcgggagtgcctcaagtatcggctcgtgggctcccaggaagaactggcgtcatggggccatgaatacgtcaggtaaggcattttctttatggacatgagaagggacttggggtcattattctgtttgtttgggaaactgaatgtattgaatcccccagacactgaattccttgactcccagcaacatgcttccctaatgagcaagtctatctatccatagacatctggcaggagaggtggccaaggagtggcaggagctggatgatgcagagaaagcacagcgggaaccactgcttaccttggtaaaggaaatcgtcccctacaacatggctcacaatgcagagcacgaggcttgtgacctgctcatggaaattgagcaggtggacatgctagagaaggacattgatgagaacgcctacgccaaggtctgcctctatctcaccaggtgagtgagtacaggagagcaggtagcactcgtcttccagggagtcttagggttcctcttgaaggaagagactttgtgactccttagtaatcctagaacatgctacattgccttctgtatagtaggcattcagtgtgtatacttgggaagggatttaaataagttacatgagttgcacgtcctgaaattgtactacttttgactttgtagttgtgtgaattatgtaccagaacctgagaactctgccctcctgcgctgcgccttgggcgtgttccgaaagtttagccgtttccctgaagctctgagattggcactgatgctcaatgacatggaactggtagaagacattttcacttcctgcaaagatgtgtatgtagagaaattggcaaaaggaaagatgagtctgtgtatgtgctttgtgtaaaaatcagagacgtagcagtttctgatgcgacacagaggcttttctgtctcatagtggctaagagtgagcaggtgtgaccgcttcccccacagggtggtccagaagcagatggcattcatgctgggtcggcatggagtgttcctggagctgagtgaagatgtagaggaatatgaagacctcacagaaatcatgtccaatgtccagctcaacagcaacttcttggctttagctcgggaggtgagggtctcctcttggtcctgaagggttttggccgaagttctgtcattctacctccctgactaatacctttcaatagactccctggttaaagttgctattcagtgggagaacagaagggacagctaggtaggggagtactggggaagcagttgtgaccctctggctttttttcagctggacatcatggagcccaaggtgcctgatgacatctacaaaacccacctagagaataacagtgagtagctctgtgggcattggagggactgttaattgttctaattgtattggGAATAACAGCTTAGACAACCCATTTCTAAAGCACTTCAAATTCTTATGGAGACATAGATTAATTTAAAGTTGGAGACTGGGCTAGCAAGATGGCTCAGTGGTTAAGAGCGCCGACTGCTCTTCCAAAGATCCCGAGTTCAAATTCCAGCAACCACATGGTGGCTCACAACCATCCGTAACGAAATCTGATGCCCTCTT
This mutation is a 1640 bp deletion beginning at Chromosome 16 position 20,654,355 bp and ending after 20,655,994 bp (GRCm38/mm10).
JAX Protocol
Protocol Primers
| Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
|---|---|---|---|---|---|---|
| 40779 | GGC AGA AGC ATC TTT CTT TGA | Common | A | |||
| 40780 | AAA GTT CCA TGC TAA GCC TCA | Wild type Reverse | A | |||
| 40781 | CTT AAC CAC TGA GCC ATC TTG C | Mutant Reverse | A | |||
| 40782 | Fluorophore-1 | CAG AGG GAG CTA GCA ACA CC | Quencher-1 | WT Probe | ||
| 40783 | Fluorophore-2 | CCC CGA ATA ACA GCT TAG ACA A | Quencher-2 | MUT Probe |
Reaction A
| Component | Final Concentration |
|---|---|
| Kapa Probe Fast QPCR | 1.00 X |
| ddH2O | |
| 40779 | 0.40 uM |
| 40780 | 0.40 uM |
| 40781 | 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.