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
>chr9:25483058+25483203 146bp ACACCAATGGGGGTCTTACC CATACCTCCAAAGGCCTCCA
Mut= 148 bp
Wt= 146 bp
Fam=Mut
Hex=Wt
Sequence
Wt Sequence (deletions in lower case):
TCCAACACTGCGGGAGTGCAAAGGGTTGATTGATTAATTTCAATAAATTTCTATGTCAATTTCTAGGTGGCCCAGCAAAAGGTAATTTTGGACACTAGCCATGGAAGATTTGATGTTTGTCTGCAGCAACTTCCTCGACCCTTAAGTCCTGTAACCTCCCGTTCTTTTGTGCCTATCAAGGCTCCCGGGTTTCTTCTCTATCCAGAGTTATTCTGTTGTGGTTGGCCATGGGGAGCACACTGGGCTGCCATCGCTCTATCCCcagagatccctcggacctgtcacataaccgcaagttcagcgctgcctgcaacttcagcaacatcctggtgaatcaggagcggctaaatatcaacactgccactgaggaggaactgatgactctgcctggggtgacacgtgcagtggcccgaagcatcgtggaataccgagaatatatcggcggcttcaaaaaggtggaagatctggcacttgtcagtggtgtaggggccaccaaactggaacaggtcaagtttgagatctgtgtgagcagcaagggtaactctgcacagcactctcccagttccctgaggcgggatcttctggctgagcagcagcctcatcacctgaccaccactgtgcccctcactcctcgagtcaatatcaacacagccacccttgctcagctcatgagtgtccggggcctcagcgagaagatggccctcagcatcgtggactaccgcagggaacacggtccttttcgaagtgttgaggacttggtgagaatggatggaatcaatgcagccttcctggacagaataagacaccaggtgttcgcagagaggtccaggcccccatcaacccacaccaatgggggtcttaccttcactgccaagcctcatcccagtcccacatctctgagcctgcagagtgaggacttggacCTGCCACCAGGGGGGCCCACGCAGATTATCTCCATGAGGCCCTCGGTGGAGGCCTTTGGAGGTATGAGGGATGGGCGACCTGTGTTTAGGTTGGCCACGTGGAACTTACAGGGCTGCTCTGTGGAGAAGGCCAATAACCCGGGGGTTCGAGAGGTGGTGTGCATGACgcttCTGGAAAACAGGTGAGTGTAGCAAGCGCAGAGGGGGTTGGGAGTGAATGCAGCACTGAGTGGCCTCAGCTGAGGATACACATGAGTGGATCCTTTGGGACAAGAAAGAGGAAAGAGTGCTTAATCCGTGTTCCTGAGGTTCCAGAGCTGATTGCCGAAAGCTGGTCCCCTCCTTAGCAGAGGCTATGTAGTGTTGGTTCT
This mutation is a 661 bp deletion beginning at Chromosome 9 position 25,482,477 bp and ending after 25,483,137 bp (GRCm38/mm10). In addition, there is a 4 bp deletion (GCTT) 166 bp downstream of the 661 bp deletion.
JAX Protocol
Protocol Primers
| Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
|---|---|---|---|---|---|---|
| 48973 | ACA CCA ATG GGG GTC TTA CC | Wild type Forward | A | |||
| 48974 | CAT ACC TCC AAA GGC CTC CA | Common | A | |||
| 48975 | GCT CCC GGG TTT CTT CTC TA | Mutant Forward | A | |||
| 48976 | Fluorophore-1 | CCT CAT CCC AGT CCC ACA TC | Quencher-1 | WT Probe | ||
| 48977 | Fluorophore-2 | TCG CTC TAT CCC CTG CCA | Quencher-2 | MUT Probe |
Reaction A
| Component | Final Concentration |
|---|---|
| Kapa Probe Fast QPCR | 1.00 X |
| ddH2O | |
| 48973 | 0.40 uM |
| 48974 | 0.40 uM |
| 48975 | 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.