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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
Mutant= 60 bp
Wild Type = 128 bp
>chr10:81490878-81491005 128bp ATCCATGCCCATAGTTGTG CTGGTCCACTGGCTACTG
Sequence
Wt Sequence (deletion in lower case):
LTCTTGCTTTGCATCTCTAGCTGCTCTGGAACTCAGAAGACTCTCCTGCCTCAGTCTGCCAAGTGCTGGGGTTAAAGGTGTGGCCACCATGCTCTGCCCAGCAGGTTCTCGTGTCTCCTTTCTACCTTAGTGCCCTGTGgctctggcaccaagtggatgggcctggcctacaggaaacctgaccaatgggtcctctgcccacagtggctgtccctgggcgcagactcaaatgggagtggcatctctgtgctgctggagctggctcgcctcttctcgcgcctctacacatacaagcgcacacatgcagcgtgagttccactaaggggcggggtccagcctaccccttgggtgctgttggccaatggaagagatggaccatctagaaaaatctgcagccagtgtctcctcccctttgttctgtggggagtcctgagccttgaaggggcctgagcagcgtcactggtctctccccatccatgcccatagttgtgacggtcacacctgtcctgaggtaccacacaggtcacatgagtaatctcagcatttgtgagctggggcccagagaggggcacaacagTGAGGCAGTAGCCAGTGGACCAGGCAGCCTCGGCATGGTCTCCAGGTGCTGCCGTGGTCACGGGACAGTAGGTCTCAGAGATGGGGGCAGAGGGGGTGTCACTTCTTTAAAAGCCATGAGGCAGCTTTGTTCTTGCTCACCCAGGATATGTTTGTCTCTTATGGCCAGCACCAGTGGCCTCACCCAGTCCTGACTGGCTTGGCA
This mutation is a 438 bp deletion beginning at Chromosome 10 position 81,490,901 bp and ending after 81,491,338 bp (GRCm38/mm10).
JAX Protocol
Protocol Primers
| Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
|---|---|---|---|---|---|---|
| 68671 | ATC CAT GCC CAT AGT TGT G | Wild type Forward | A | |||
| 68672 | CTG GTC CAC TGG CTA CTG | Common | A | |||
| 68673 | AGG TTC TCG TGT CTC CTT T | Mutant Forward | A | |||
| 68674 | Fluorophore-1 | CAC ACA GGT CAC ATG AGT AA | Quencher-1 | WT Probe | ||
| 71782 | Fluorophore-2 | ACC TTA GTG CCC TGT AGT GAG G | Quencher-2 | MUT Probe |
Reaction A
| Component | Final Concentration |
|---|---|
| Kapa Probe Fast QPCR | 1.00 X |
| ddH2O | |
| 68671 | 0.40 uM |
| 68672 | 0.40 uM |
| 68673 | 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.