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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
>chr5:134196569+134196669 101bp TGGAGAACAAAGCAGGGATCT GCTGCCATACTTGGCTCAG
Mut= 106 bp
Wt= 101 bp
Fam=Mut
Hex=Wt
Sequence
Wt Sequence (deletion in lower case):
CAGTGTCAGCCGTCACAAGAGGGATAGAGCCATCATGGCAGGAGAGCCAGCAATGGGCAGAGAAAGGGTGGGGCCAGGAGCGGGAAGCTGGCTGGTCACATTGCAGCCACACGCAGGAAGGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAAAGAGAGAACGCGCAAGCGCGTTAGTTCTTTGGGCTCTTGATGTATGTTCAAGCTACATGCTCCTGTGTCTATTGCCCTAGATGAAGCtggtggtagaaagtagttctgagagtcccagttaccatctcttacctcctgtcctaggtaaagccttggggacaacagccatggtccctgttccctacgagcagatgctacaggatgaggcagctgtggtggttcggggccttccggaaggcctggccttccagcacccagacaattacagccttgccaccctgaagtggatcctggagaacaaagcagggatctccttcgctgtaaagaggtaatctgctccggagCCTGACAGCCTGTTTAGAGAGAAACTGGCCTGAGCCAAGTATGGCAGCACAGGCCTGCAACCTCGGTACCGGGAGACTGAGACAGGAGAGTCACTGAAAGTTTAAGGCAAGCCCAAGTTACAGTGAGTTCAAGGACAGCCTGGGGTATATACCTAAATCCTGTCTTTTTTCTTTCTTTCTTTCTTTTTTTTTTTTTTAAGATTTATTTATTTCATGTATGTGGGTACAACTGTCGCTGTCTTCAGACACACCAGAAGAGGGCATCAGATC
This mutation is a 257 bp deletion beginning at Chromosome 5 position 134,196,365 bp and ending after 134,196,621 bp (GRCm38/mm10).
JAX Protocol
Protocol Primers
| Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
|---|---|---|---|---|---|---|
| 47737 | TGG AGA ACA AAG CAG GGA TCT | Wild type Forward | A | |||
| 47738 | GCT GCC ATA CTT GGC TCA G | Common | A | |||
| 47739 | TGG GCT CTT GAT GTA TGT TCA | Mutant Forward | A | |||
| 47740 | Fluorophore-1 | AAA GAG GTA ATC TGC TCC GGA | Quencher-1 | WT Probe | ||
| 47741 | Fluorophore-2 | CCC TAG ATG AAG CCC TGA CA | Quencher-2 | MUT Probe |
Reaction A
| Component | Final Concentration |
|---|---|
| Kapa Probe Fast QPCR | 1.00 X |
| ddH2O | |
| 47737 | 0.40 uM |
| 47738 | 0.40 uM |
| 47739 | 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.