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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
Mutant= 130 bp
Wild Type = 77 bp
chr4:134836179-134836255 77bp GGTTCCTTTCCCCAATATTATC CAAACAAAACCCCACAAATC
Sequence
Wt Sequence (deletions in lower case):
TTATTGGTAATGAATAGTGGTGTCCAGAGGAAGGCAGCATTATTAATATTACCACCAGCATATTTCTTGCCAATATTTTGGTTCATCTGCATTCCAGCAAGCTTTAAGAATGAAGCTCTTTAGGAATGGTTTATGAGGAAACAGGAAGCTTTTCAGTGTTAAAGTTCTTGTTCATTCAGGTAAAACCTGCCTATAGAATCCCAATGAATTCACTGTGGTCCAACCTCAGTTTCGGCCCAGTTCATGTCCTTGGCTTAAGAACCATGAACAAGAGCACTCTTTAATGTTGAATTAGCTACTGCTTTTTAGTATATGTGCTGCTCAAGCAAGCTTATAACCActgcttccccctggtctattctgagtgtttgaaatttcctcttgttaaattgctttgtattcagcaattttgttcaacaaagcttttaatagcatacttgaaattaacaaattctgaaatgtggcttttaatttggttttttttttctgtctttaggccttctcagtattttttgtttgtgtagcattcacttcaaatatcatatgtctcctcttcattcccatacaatggctttttttcgctgctagcacatatgtatgggtccagtacgtatggcacacaggtgagtcttaatgacttcttggttaggaattttagattcaagttaattatttagattgatattttaaggttcctttccccaatattatcttaatatgttgcttggcttgtggttgtggtttgttgatttgtggggttttgtttgttctttggttgtaggattggttatttgaggcagggtcttactagtaatcctggttgtcctgtaactcactatgtaggcCTGGCCGGTGTGGAACTCAGAGTGATCCTCCTGCCTCTGCCTTCTGAGTTCTGGAATTACAGCTGTGCCTTGCCTTACCCTGCTCTCGCTTGCATGCATGGTTTCCTTCTTTTTGATCTGAATGAAAAGCTTAGGTGTTAGGTCTTCTGGTTGGTCTCTAGTAAAGATGCCAGGTTAAAGGGTCAGGGCGGCTTCTGTTTAGGAGGCCTTGAAGACATTCGTGTGCCACACAAGACTGTTTACAACATTTTATTATTGAAAGGAATGCTAGTTAG
This mutation is a 505 bp deletion beginning at Chromosome 4 position 134,563,412 bp and ending after 134,563,916 bp (GRCm39/mm39).
JAX Protocol
Protocol Primers
| Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
|---|---|---|---|---|---|---|
| 59011 | GGT TCC TTT CCC CAA TAT TAT C | Wild type Forward | A | |||
| 59012 | CAA ACA AAA CCC CAC AAA TC | Wild type Reverse | A | |||
| 59013 | Fluorophore-1 | ATG TTG CTT GGC TTG TGG TTG TG | Quencher-1 | WT Probe | ||
| 59014 | TGG CTT AAG AAC CAT GAA CAA G | Mutant Forward | A | |||
| 59015 | GCA GAG GCA GGA GGA TCA C | Mutant Reverse | A | |||
| 59016 | Fluorophore-2 | AGC TTA TAA CCA CTG GCC GGT G | Quencher-2 | MUT Probe |
Reaction A
| Component | Final Concentration |
|---|---|
| Kapa Probe Fast QPCR | 1.00 X |
| ddH2O | |
| 59011 | 0.40 uM |
| 59012 | 0.40 uM |
| 59014 | 0.40 uM |
| 59015 | 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.