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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
>chr2:26008521-26008660 140bp CCAAACTCAGGAGCTCTTCATC CACACACCCCAAAAGATCATA
Mut= 145 bp
Wt= 140 bp
Fam=Mut
Hex=Wt
Sequence
Wt Sequence (deletion in lower case):
GGTTCTTTGTTAGTTTTTGAGACAATAGCCTCACTGTGTAGACGAGGCTGACCTTGAATTCACAGAGATCTGCCTGCCTCTGCCTTCCAGCTCTGAGATTAAAGGTAAGGCCCAATATGCCCTTGTAGATTTGGAGGGCTATGTCTGGGATAGTGTTGCAGATTTAAgcaagtttgttggacattctgagaaataatctttttggtttgtttgatttttgagacaggtttttactgtagaattaaggttggcctcaagctcccagcagacttcttttctcagcctcgcaagagcagggattacaggtgtgcatcactgtaccgggcatgatctctttctttatcctactgcagttccagacagaactccggaagatcctcgtgtctctcattgaggtggcacagaagctgttagcactgaatcccgatgcagttgaattgtttaagaaggcaaatggtatgcgtgggtcctaggctgctcctaaaagtgtccccagccctcacactgaaagcaagttctgctttggttaatgggggagagggtttggtgtcagtaattgctaggtgaggtgagcctctactgatgaggctggggggcttcaaaggactctggaatcccctcatggcaggccttgtctttctcctcagctatgctggatgaagatgaggatgagcgagtggatgagactgccttgcggcagctcacagagatgggctttcctgagagcagagcctcaaaggcccttcggctgaaccagtatgcttgctttcttcttgccctgggcttccctgtgattctccttccaatctctggagtcatacttttttttttaatttttatttatctgtgtatatgtacatgtgtgtatgggtgcccatagaagccagagaggaagttggatcttctggagctagaattacagactgctgtgaactgcctgacacaggtgctaggaaccaaactcaggagctcttcatcttaatctaggagccagctccccgatcccattgagtcatttctaattgggtgttctagtagagGCACCTTATTGCTGTGCTTCAGAAGTGCTTTCATATATGATCTTTTGGGGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGGAGGGTCTCTGTTACCTATGCTGGACTTGAACTCCTGGACTTATGTAGTCCTCCTGCCTTAGGCTCCTGAGTAGCTAGGATTGAAGGTGTGAACCATGATACC
This mutation is a 873 bp deletion beginning at Chromosome 2 position 26,008,577 bp and ending after 26,009,449 bp (GRCm38/mm10).
JAX Protocol
Protocol Primers
| Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
|---|---|---|---|---|---|---|
| 46095 | CCA AAC TCA GGA GCT CTT CAT C | Wild type Forward | A | |||
| 46096 | CAC ACA CCC CAA AAG ATC ATA | Common | A | |||
| 46097 | TCT GCC TTC CAG CTC TGA | Mutant Forward | A | |||
| 46098 | Fluorophore-1 | CCG ATC CCA TTG AGT CAT TTC | Quencher-1 | WT Probe | ||
| 46099 | Fluorophore-2 | TGT TGC AGA TTT AAG CAC CTT ATT G | Quencher-2 | MUT Probe |
Reaction A
| Component | Final Concentration |
|---|---|
| Kapa Probe Fast QPCR | 1.00 X |
| ddH2O | |
| 46095 | 0.40 uM |
| 46096 | 0.40 uM |
| 46097 | 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.