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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
>chr5:105891106+105891202 97bp CACAAATTACAGCATATGGGT CAGTAGCAATCGTCACATTG
Mut= 91 bp
Wt= 97 bp
Fam=Mut
Hex=Wt
Sequence
Wt Sequence (deletion in lower case):
TTAGTGAAATAAAGGTTTCTTCTAAGGTCTTTGATGGGAAAACATCAAGTAGGAACTCTGAAGAGGAGTTGTCAGAGAGTTGTACAAGTGGTGACCTGACAGGCACACGGTGGTGTTTATGATCTCTAACAATTTTGAACTGTAAAAATTTTTTAAAAAAGGACATACCCACCCATgtaatattggaaattctaatttattggaattaagttcatgtaataatttataaaatttgtaaatgtgctttctcagagtggacatgtaaaattcagttacgttccacagaattttcaggtgaaaactagtaaataggcgacaaacgaattgtattaattcatagttaatctattgacattatgctaatgttttttgatttcacaaattacagcatatgggtgattttggaagcttccatagacctggaattattgttgactatcaaaacaaacctgccaatgtgacgattgctactgcaagaggaataaaaagaaaaatgatgcaaatatttattaagcccgggggagcctttatcaagaagcctaagctagcgaaacctatggataagatgaacctcagcaaatcacctagtaagtaagactattgctcgggacgcttcatcctattagaacacactgatcTGTGGTGACTCTTAGTGTTCACATAGAAAAGTTATAGCATGAGTTATAGCTGAAAATAACTGCGAACCCCTCATGTAGTTATTAATCGATTGTTACTATTCAAACGTATGTTTGACTGCTTTGTATGTATGTATGGATGTATGTATGTACGTA
This mutation is a a 460 bp deletion of Chr5:105,890,907-105,891,366(GRCm38/mm10).
JAX Protocol
Protocol Primers
| Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
|---|---|---|---|---|---|---|
| 73778 | CAC AAA TTA CAG CAT ATG GGT | Wild type Forward | A | |||
| 73779 | CAG TAG CAA TCG TCA CAT TG | Wild type Reverse | A | |||
| 73780 | Fluorophore-1 | AAG CTT CCA TAG ACC TGG AA | Quencher-1 | WT Probe | ||
| 73781 | AAA AGG ACA TAC CCA CCC AT | Mutant Forward | A | |||
| 73782 | GGG TTC GCA GTT ATT TTC AG | Mutant Reverse | A | |||
| 73783 | Fluorophore-2 | CTG TGG TGA CTC TTA GTG TTC ACA T | Quencher-2 | MUT Probe |
Reaction A
| Component | Final Concentration |
|---|---|
| Kapa Probe Fast QPCR | 1.00 X |
| ddH2O | |
| 73778 | 0.40 uM |
| 73779 | 0.40 uM |
| 73781 | 0.40 uM |
| 73782 | 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.