For in-depth product & services help, ask our
Technical Information Scientists
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
>chr16:29219049+29219137 89bp AGTGACTCAACAGGAAAGAGCT GCTCACAACGGGCCAATTTA
Mutant= 81 bp
Wild Type = 89 bp
Sequence
Wt Sequence (deletion in UPPERCASE):
taagcaccattgtaaagaaatttgcaggagcttcccctcctacccttactgctaacacacacacacacatacatacacacacacacacacagagagagagagagcactgtgagtctgagttgttaccagaggtcatctcggtaatttctttgtccctCTGATAATGTGGAACCAACCCTTGCTCAGCTTCTCATCCTGTGAATTGGAACAGCAAGCTCATCTTTATGCCTGTCCTTACAGATGGCGGTAAATGATTGCTTCAGTCTGACCTATCCTCACAACCCACACCCAGGAGACTTGATTGAAGTGTTCCGTCCTTGCTATCAGCACTGGGCACTGTACTTGGGTGATGGCTACGTGATCAACATTGCACCTATAGGTAAGATTTGTTTCCAGGGGCCCCCTGGGAGATTTTAACTTGTTTTGTATTTAGTAGCAATCACAGATAAGATCATAAAGTAGATAAAATACAGAAACTAAATCCTACTAATTTAGAAAATGATATTCAAAGACCAAAGCGTCATTTAAAAGATTCCACAGACATTAAAATTAAAATCTCTAAAAAATTCCTAATGATGACTTGAGGGAGGCATTGGCCCCAAACATGACTAGACTCTGTGTCCTTATGTCTCACACATGTGGGTGATTTTACTTAATTCAGCAGCTACTCGTAGGTTGTGTAGTATGTGATGAATGCCTCTCTGGGTGTTGCGGCTGCCCAGCTATATAGATATCTACACAATGGTACTTATACTTTCACAGACAGTGGGAGAAAAGAAGAACAAATTATTAGACGTGCTAGGAAGGATGTAGAAAATCCAACTGAGGAAGGTGGCTGGTGAAGCCCTGGATCTAGGAAGAAAGGAGTGGGCAAAGGGGTTGAGGGGTGTGTTACAGTTAGGAGAGTTCTGCAAGGAAGCAGCATCAGAAGATGAGAGAAAACTAGTTCCATTGTCCCTGGGAAACTCGTGCCTCAGAGGAGAGGCAGCAAAAGCTAAGAACACAAGCCTCGAAGTGGGAAGCTAAAAACAGTTGACAAAAGAAGAGAACAGATATTTCAACTTTATATCCACCGTTATAGATTGAGACACGGACTGATCTCTCACCCATCTCTTTAGCAAAGGATAGAATGATAGTGTGCAGTGTGGGCAGAGACAGGGGAACTGGGTACAAGGATATGGTTGGTGGAAATAGAAACTGATAATGCCATTCACAAAACCACTTTCACAGTATGTGTTAAGTGTTTTCAAAGATTTCTTATACTCTGCATCTAATTTTACTTCCAGGAGTCTGGGGAAACATTTAAAAATGTAGACAAATTTCATGTCTAATGAAATTTAAGAAAACGAGACAATGGCTTAATTTTCAACAGGGGGCTGGTTAAGCACTTTATGACTTAGCAAGTAGGTATGAGGTATCTGTGTCTGTTAGAATCCTTGAGTTGTATATCACAGAAATAAAGCTAACACTAGCTTAAGCAAACAATCAAAAAGACAGGTCCATGACTTACATCAGAAAGGCTTGAGCTGGCCTTGGACAGCCTCAGGGTTAGGAATGTCATCACATCAGGACCTTCAGACTCACATGTGTCTTCCTTCTCCTGCCTGTTGCTGTCATTCTTTGACTGTGGAAAGAAACAGAGACAGCTCACAAGCTGAGGCACCATTCTTACAGTTCAGTGACTCAACAGGAAAGAGCTAATCTGCTCTCTCTGTGTTGGAGTTTAGAAATCATGGAGAATGACTCTAAATTGGCCCGTTGTGAGCCACAACTCCATTTCTGAAGAATAAGATGTAATACAGTGATTGGCTCTGGCAGGCAGTTTGAGGAAGAAATAGGCAATGGTGGAACTATAATAAGATAAAGCAACTATAATAGCAATTTTAAAAGCACACTTTTAGACATGTTTAACTATTTGGGAGATGGTCTTAATGTAATATTTTAAAATGTCTAAAGTGCAATATGTGACATAAAACTTCACTTAAAAGAAGTGTGACATATATCAATGTATACAACATCTGTTCCTGTGTACTTCCAGTGTCTATAATATAAACTGATGAATGTTGCTGCTCTGTTGAAGAGACTGAGATAGAAGAGGAATGGAAAACGATTTTGATTTAAGTCAAAACTGAAAGAGAAACTTACATGGCAGTTTATGTAGAGTTCCTTCCTCCTAGGAGTTACAGGAATATGATATGCATAGAAGGTAGGCACATGTGGAGAGAGAGCAGGTCCCAGAATATGCCAAGCCACAAGGAGTCATGGTACTGGCATCATGGGCCCAGATACAAAGGGTCCTGGCCATCTAGGCTGTGATTATCAGTAGAGGTATCTCAGGCGAAGAGACTGAAGGATTCTTCAGGCAGGGAAAACATAGAATGGGGCACTATCTGTGTTCTGCCTACTGAAGTTAGCTTCAGGTTTGTGACATTAGTTTGCTGGCCAGAGGAAATGACAGAAAGAAGACAGGATCTTCCAGCAGGAAATGTGGGCTTCCGTTAAAGCCCAGGGAAGAGCAAGTGTATGAAGAAGGAAGAAGTACAGAGAGGTGGTAGAATCAGAACACAGCACTCCTAACCCCTAAGATATCCATTTGGAAGAGTTCAGATGCTAAGAAGGAACAGGCACAATAGAATAATTGGTCAATAATCTTAATCCTACTTTGTTAGAAATGTATGCAAAAAAAAATCTCTCATAATACCAATCATTAAAGGCCTTACTTCCCAAGAACAGGCAGAGAATTCCATGCAACTTGTATTTTTTCTCATATGTAATTGAGAACACAGTGTGTGGTTGAGATCAAGCACTTTTAGTTTACTGCCTAAGGAGAAGAGAGTAATTGAAGTTCCCCCAGCATCTCTAGCATTCATTCCTAGATGGCATTGGTACTCTGTGGACCAGATCGCTAAGCCCCACCAACCAACGTTCTCATTACAGATGGCATTCGCTCATCATTTACAAGTGCTAAGTCCGTGTTCAGCACAAAGGCCTTGGTGAAAATGCAGCTTTTGAAGGATGTTGTGGGAAATGACACATACAGAATAAATAACAAGTACGACACAACATACCCTCCTCTTCCTGTGGAGGAGGTGATACAACGGTCAGAGTTCGCTATTGGGCAGGAAGTAGCCTATGACTTGCTGGTCAACAACTGTGAGCATTTTGTAACCTTGCTGCGCTATGGAGAAGGAGTGTCAGAGCAGGTGAGTTTTCTTTTAAAGGTTCAATAAGTTTCTCACACTGAGAAAATCCTATCTGTCATGATAAAGGTCCTACACTTCAACCAAAATGCAAAGAACAAGAAAAGTTGGAAACACCAAAGGGCAGAGAAGGAGGCTATTAAAAATATAGTAAGTTAGAGATCAAACAGACTAAGACTTGGACGTCTTGAAATCTGGGctggaaggggacgtgtggtagcatcaaatccatagcttcctgtcagtcttactgaccccactagtcacagttttcattactcccccattcagcaacctcaaaacaaccacgccatgtgcagaaaataatggagcaaaaaaggaagacaaatgggttaattagtagattctttcccagtagggaatagcggccaaccaatggcatgccttgaagggttggtaaatgtttcctatttctttacaatgtg
This mutation is a 3242 bp deletion beginning at Chromosome 16 position 29,217,529 bp and ending after 29,220,770 bp (GRCm38/mm10).
JAX Protocol
Protocol Primers
| Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
|---|---|---|---|---|---|---|
| 67773 | Fluorophore-1 | AAT CTG CTC TCT CTG TGT TGG AG | Quencher-1 | WT Probe | ||
| 68576 | AGT GAC TCA ACA GGA AAG AGC T | Wild type Forward | A | |||
| 68577 | GCT CAC AAC GGG CCA ATT TA | Wild type Reverse | A | |||
| 68578 | CCA GAG GTC ATC TCG GTA ATT T | Mutant Forward | A | |||
| 68579 | AGA CTG ACA GGA AGC TAT GGA | Mutant Reverse | A | |||
| 68580 | Fluorophore-2 | CTT TGT CCC TCT GGA AGG GGA C | Quencher-2 | MUT Probe |
Reaction A
| Component | Final Concentration |
|---|---|
| Kapa Probe Fast QPCR | 1.00 X |
| ddH2O | |
| 68576 | 0.40 uM |
| 68577 | 0.40 uM |
| 68578 | 0.40 uM |
| 68579 | 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.