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
>chr3:152128442-152128529 88bp CAGGGGTTTTAGAAGGATGTGA CAACCCAGTCACCTAAGAAGG
Mut= 82 bp
Wt= 88 bp
Fam=Mut
Hex=Wt
Sequence
Wt Sequence (deletion in lower case):
TGCTGCCCCTCCGGGGGACATGTTTGGTTTCCAGCACTGCAGCCCTCTCTAACTTCATCTGCAGGGGATCTGATGCCCTCTGTAGATTCTCACAGGGGTTTTAGAAGGATGTGATGTGGCAGGTGTGACAGGTACgcagagggaagaacagtgtctcacccttcttaggtgactgggttggaggatgctggggaaagatgtctagaggaaatggtagctgtgagctagctgaattctgaatggaaggaagatgaagaataggtggagatagggcgtttgttctttctcgcttcaccttctgtccctaagtgagcattaaactacattgtaaatatgtcagcaggaacacaagaggtaaagccacacagttccacatgatatcaccattgattttctctctttcagagaattaaagatggcagcaccattgactcagttaaaacgatctgtgtgggggatcatatcgaatgtataaacggagaaaatattgttgggtggcgtcactttgaagtcgctaagaagttaaaggaattgaaaaaggaggagctctttactttgcagttaatagaacccaagaaggcttttggtaagttgagcccgtgtgtggtgtgtgtgtgtgtgtgtgtgtgtgtgtgtgtgtgtgtgtgtgtgtgtgagagagagagagacagacagagagagagagagagagagagagagagagatatggatggacagacagacaaagagaaaccattttcttccccttgtttatctaagaggcaggatactgCTCTGTGGGGCTGTGTGCTTTTAGCATACCATCTTGTCTACTGTACTGTACTAGGGAAGAACACAGAATATTACAGACAGCACTACTTAGAAGGAAAGTAACTCTGAAGGGAAAGGAAAGTTTTCCATTGAAGAACCTTTTTCTAA
This mutation is a 637 bp deletion beginning at Chromosome 3 position 152,127,850 bp and ending after 152,128,486 bp (GRCm38/mm10).
JAX Protocol
Protocol Primers
| Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
|---|---|---|---|---|---|---|
| 45425 | CAG GGG TTT TAG AAG GAT GTG A | Common | A | |||
| 45426 | CAA CCC AGT CAC CTA AGA AGG | Wild type Reverse | A | |||
| 45427 | AGA CAA GAT GGT ATG CTA AAA GCA C | Mutant Reverse | A | |||
| 45428 | Fluorophore-1 | CAG AGG GAA GAA CAG TGT CTC AC | Quencher-1 | WT Probe | ||
| 45429 | Fluorophore-2 | ACA GGT ACC TCT GTG GGG C | Quencher-2 | MUT Probe |
Reaction A
| Component | Final Concentration |
|---|---|
| Kapa Probe Fast QPCR | 1.00 X |
| ddH2O | |
| 45425 | 0.40 uM |
| 45426 | 0.40 uM |
| 45427 | 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.