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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
>chr5:90222311-90222405 95bp CTGAAGCTAACCATGCAGTAG TTCTGGTTGCAAGTTTTCCA
Mut= 90 bp
Wt= 95 bp
Fam=Mut
Hex=Wt
Sequence
Wt Sequence (deletion in lower case):
GGGGCTGGTGGCACACAGTGTCCTGTGTTCTTCCATGTCAATTAGTGATTAAAAAATGCCCCATAGGCTAATCTGATGGGTGAAATGTCTTAGTTGAAGGCCCTTTCTTCTGGATGTGCCAAGTTGACAGCTGAAGCTAACCATGCAGTAGTTATCAGTAAAGATCCTTTAtgaactcatcaaacaacttttttctttagttaggtggaaaacttgcaaccagaaataaaagacatcgccaagcgccttaaccaagaagttgcagtctgtgcccgtcagtttggatggtccaagagagtggctaggtatgcagctttgggtctgcggtcacgaaaatgtgtgtgtggtgtgtgcgtgtatacacggttaggtattcttttgattggcgtattcatattaatcactatgctgcATGGTGGTTTTATGTATTGGAATGGAATCAAAGAAAGGAAGAAACAGGCGTTATTCCAACTCCTGTGAAGCTGAATCGTCCTCCTATCTCAGCCTCTCTCTCAGCTCTGAGATTACAGGTGCAGACCATTGTATCTGGCTCTGTATGTCAGAATATGACAGGGTTTCATGTGCACCAGGGATGCCCAGTGG
This mutation is a 241 bp deletion beginning at Chromosome 5 position 90,222,124 bp and ending after 90,222,364 bp (GRCm38/mm10).
JAX Protocol
Protocol Primers
| Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
|---|---|---|---|---|---|---|
| 45575 | CTG AAG CTA ACC ATG CAG TAG | Common | A | |||
| 45576 | TTC TGG TTG CAA GTT TTC CA | Wild type Reverse | A | |||
| 45577 | GCC TGT TTC TTC CTT TCT TTG A | Mutant Reverse | A | |||
| 45578 | Fluorophore-1 | CAT CAA ACA ACT TTT TTC TTT AGT TAG G | Quencher-1 | WT Probe | ||
| 45579 | Fluorophore-2 | AAG ATC CTT TAA TGG TGG TTT TAT GTA | Quencher-2 | MUT Probe |
Reaction A
| Component | Final Concentration |
|---|---|
| Kapa Probe Fast QPCR | 1.00 X |
| ddH2O | |
| 45575 | 0.40 uM |
| 45576 | 0.40 uM |
| 45577 | 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.