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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= 100 bp
Wild Type = 114bp
>chr9:121965198+121965311 114bp CATGGTTGTCCCACTCACAG GCTCCTGGCAAAGGATCTCTAC
Sequence
Wt Sequence (deletion in lower case):
AAGCTCTGGAGTAGGGGTGGGTGCAGCTGGCTGGGCTGTCATGTTCCCCTGTGGGCAGCAGCTAGGTGTCCTTTTGCTGCTGCTTCCTGTCCCCCCATACTCTGACCACTGCCACTGCTGTTCTGTGTTTTAGAGATGATGGCTTTCCTAGGTCTCACACTATCTTATTTACCCCGTCCTCAGGTGTCTTGGATCTGGAGAAGGCTTCGTGGCAAGAAGCGGTCAGTGATGGCattctgcctcttgatggtcctgtctgcagtggctgtcattcatttccctccagggcatccagcttccaccccaggacttaatcccatggagcctcggggggaagttggtgcgtctgaccccaggattcagcagactttgaactctagcctgaggcagccagcaaggaacctgggccactggacaggccaggctttgcccagaaatcccatcttggtctgtgccaagaagcaaagccgcagaaggcaagtggacagaagccgacacccactgagtgtcaggagagatgcgatactcagtgcccaggatcgtgagcttctattagagggggaggtgagagatgcagggggcgcagcgctgggccagcctgggcataatggtttagtccaagagacacagagcaagacagtcaccatggttgtcccactcacagagagaagccacgaatcttttcaggcacagagagatactgctgcagcaagctttaggccatggccagcagatggtagagatcctttgccaggagccaagaatggagtcttgactggtgggaaggctgggagtgccacctcagggtcagaggctccatggtggtctagctctgctgaggacctgcagaaatccccatggtgtggtactgagacccctgggctggccggcactgcagcatggggtcaggttccaccatggttcatggaacatgatgcacaaactctccggctactggtccatgggaaagtggtgggaaaagccagggtccctgcccatgggcaggtgctgcaggttggactatctgctggagatgccctccaggacatatctcctctcaggctcagccagttctgctcccagggtctctgtggcctgattaagaggtccaaggacctgcatgaagtcctatccttccacctggaccgtgtgctgggactccaacggagcctacctgccgtagcccggagcttccacagtccccttctgccctaccgttacacagatggcagtgtaaggcccatcatctggtgggcaccggatgtgcagcacctaagagacccagatgaggaccagaactctctggccctgggctggctacagtaccaggccttgctggcccgaggctgcaaatggccaggccagatcccatgcctgggcatccaccatgctgAGTGGGCACGTTTGGCTCTCTTTGACTTTCTGCTTCAGGTAGGTGTGGTTGGGCAGTGGGGTGGAATGCAGTGGAGTTAGGGGGTTCCAGGTGAAGACCAGGATGCCCAGCCAGGATGCCTACTCCCCACAGTGCTAGACTTTTGACCAAGCAACTGTTCTTATAATGTTCCTGCCTGTTTATCCTGTGGTACCCTAGAGCAGGGATGGTCTATCCTTTGGTACCCTAGAGCAGGGATGGTCTATCCTTTGGTACCCTAGAGCAGGGATTTTTCTCTCCTGTGATACCCTAGAATAG
This mutation is a 1160 bp deletion beginning at Chromosome 9 position 121,964,787 bp and ending after 121,965,946 bp (GRCm38/mm10).
JAX Protocol
Protocol Primers
| Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
|---|---|---|---|---|---|---|
| 67892 | CAT GGT TGT CCC ACT CAC AG | Wild type Forward | A | |||
| 67893 | GCT CCT GGC AAA GGA TCT CTA C | Wild type Reverse | A | |||
| 67894 | AGG TGT CTT GGA TCT GGA GAA G | Mutant Forward | A | |||
| 67895 | CCA CAC CTA CCT GAA GCA GA | Mutant Reverse | A | |||
| 67896 | Fluorophore-1 | CAG GCA CAG AGA GAT ACT GCT G | Quencher-1 | WT Probe | ||
| 67897 | Fluorophore-2 | TCA GTG ATG GCA GTG GGC A | Quencher-2 | MUT Probe |
Reaction A
| Component | Final Concentration |
|---|---|
| Kapa Probe Fast QPCR | 1.00 X |
| ddH2O | |
| 67892 | 0.40 uM |
| 67893 | 0.40 uM |
| 67894 | 0.40 uM |
| 67895 | 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.