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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= 118 bp
Wild Type = 154 bp
chr4:141193328+141193481 154bp GCCTACCAAAGATCACATTTCC CAACACAGAGAAAGGTACTCAGC
Sequence
Wt Sequence (deletions in lower case): TCATGGAGCCCATGCTGCTCTCAGACTCTGTGTGTAGTTGAGGCTGCCCTTGGACTAAGTTATGACAGGGTTATACCCCACGGGACCTGTCACTTAATACACCTCCCGGAGACCACAGCACAGCGACACAGAATGGCTTGTAGTGTCCACCGTTCTTTCTGTGACCGTGAGACGTACTTGAGGTGATAGTCCTCTGTTACCTGACATTCTCACTAGCCTACCAAAGATCACATTTCCTAGGAGAATCCCACTGCCTGTGGGCCATTGTGGCACTGTtgtaaagttaagccaaggcaacttgggaccttttgaattgggtgcccattccactccactttcaaagaaagctgagtacctttctctgtgttgggtatgaagatgtataaatcaataatgggtctcttagacctgaaaacccagctccccgccccccccagcccccttacctttctgctgtgtgacagacattgcctctcttcttgtccttcccaggaaaggtgacctgtgggtgagctcccaaatcctaatcacggtgctgttgccttttcctagggtcctatccttcccccaaagctggggccactctggtcgtgtacaaggacttgctagtgctatttggtggctggacaaggccaagtccctatcccttacaccagccagagagattctttgatgaaattcacacgtactcgccgtctaaaaactggtaagccccaaagagaacccagTCTGTGTCTGGAGGAAATGGGTTTCATTGTGTTATGCTTTAGCTCTTTTGGGTGTGGTGGGCAGAGGATGGACGTCCGGAGCCTTCTCTCTTCTCGCCATGAGAATGCTAGAGATCAGTTCACTTTGTCAGGCTCCGTGGCAAGTGCCTTAGCCCACCAAGCCAGCTCGCTTCCCCCAAAGTGGGCTTTAAATCGATGGATTCTTTATTTCTGTGTATCAAGAAGCTCGCGG
This is a 452 bp deletion beginning at Chromosome 4 position 140,920,700 bp and ending after 140,921,151 bp (GRCm39/mm39).
JAX Protocol
Protocol Primers
| Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
|---|---|---|---|---|---|---|
| 59539 | GCC TAC CAA AGA TCA CAT TTC C | Common | A | |||
| 59540 | CAA CAC AGA GAA AGG TAC TCA GC | Wild type Reverse | A | |||
| 59541 | Fluorophore-1 | AGC CAA GGC AAC TTG GGA CC | Quencher-1 | WT Probe | ||
| 59542 | CCA CAC CCA AAA GAG CTA AAG | Mutant Reverse | A | |||
| 59543 | Fluorophore-2 | ATT GTG GCA CTG TTC TGT GTC TGG | Quencher-2 | MUT Probe |
Reaction A
| Component | Final Concentration |
|---|---|
| Kapa Probe Fast QPCR | 1.00 X |
| ddH2O | |
| 59539 | 0.40 uM |
| 59540 | 0.40 uM |
| 59542 | 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.