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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
>chr2:181308518-181308634 117bp CTGAGCAATTGGAATGAGATG ATTCAACCTGCCCTCCCTCT
Mut= 120 bp
Wt= 117 bp
Fam=Mut
Hex=Wt
Sequence
Wt Sequence (deletion in lower case):
AGATGGAAGCAGACAGATAAAGAATTCAAAGTCATTGTAGGCTACAAAGTGAATTTAAGGCCAGTCGGATTACAGGAGACCCCTTCACAACACAAAACAAATATGCACATAGATAAAGGCCACTGTAGAATACAAAGACCTAGAAATACcacaggatcatggtacctgctcaccttgactttacatccacagcctacaaggagaagatgaaggagttatctgtgctgtcgctcatttgctcctgcttctactctcagcctcacccgaacaccatctaccagtatggaggtgagtgtatccacacagctgctggccctgctcagctagggcctgacaaccttacttctctcgtttcttctggcctctggcaatgctgtgaactggctggagagagtcattctgggaggaggggcctatgctaaagagtaacggcaccccttcttaacccttgttgggatgtgtgctggaaataaacctctcatcctgcctgtatgcccaaaccaggcctcctgaagtcccatctggtcttgtcctacagatatggaagtgaagcagctggataagcgggcctctggccagagcttcgaggtcatcctcaagtctccttctgacctatctccagagagccctgtgctctcttctcctcccaagaggaaggatgcttccttggaagagctgcagaagcggctggaggcagctgaggagcggaggaaggcaaggactctccttacacacttgacattgctgaccaggccctgtgcttggtcagggcattgggctgggtagtgcagaacttactgctgagcaattggaatgagatgaaagatggatgtgccaagatggtaacaaacacagggcaataggaacCACATTCTTGAGGGGGACCTCGATCATGGCAGAGGGAGGGCAGGTTGAATTCCCCCAGAAAGAGCCCTTAAGACATAAAGCTGAGGCTAGTGCTCCTTTCAGGACAGAAGATCGAGGAGAAAAGGTCTCACATCTAAAGACAGTTAGCTGGGTTGGGTA
This mutation is a 718 bp deletion beginning at Chromosome 2 position 181,308,568 bp and ending after 181,309,285 bp (GRCm38/mm10).
JAX Protocol
Protocol Primers
| Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
|---|---|---|---|---|---|---|
| 47011 | CTG AGC AAT TGG AAT GAG ATG | Wild type Forward | A | |||
| 47012 | ATT CAA CCT GCC CTC CCT CT | Common | A | |||
| 47013 | CCC CTT CAC AAC ACA AAA CA | Mutant Forward | A | |||
| 47014 | Fluorophore-1 | ATG GAT GTG CCA AGA TGG TAA | Quencher-1 | WT Probe | ||
| 47015 | Fluorophore-2 | AGG CCA CTG TAG AAT ACA AAG ACC | Quencher-2 | MUT Probe |
Reaction A
| Component | Final Concentration |
|---|---|
| Kapa Probe Fast QPCR | 1.00 X |
| ddH2O | |
| 47011 | 0.40 uM |
| 47012 | 0.40 uM |
| 47013 | 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.