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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
>chr5:21781122-21781223 102bp GGGACCTTGAGTGTTCTCAG AACCCAAAACAAACGAAACC
Mut= 97 bp
Wt= 102 bp
Fam=Mut
Hex=Wt
Sequence
Wt Sequence (deletion in lower case):
ATCCACGACTGGCGCCTAACTTACATGACTGCACAATGACTTACATTCTTTTTCTTGTTGGTTGGTTGGTTGGTTTTGGTTTTGTTTTGTTCTTTTGATATGGGGTTTCTTGGTGTAGCCCTAGCTGTCCTGGAACTTGGATCTGTAGACAAGACTAGCCTTGAACTCATAGAAATATTTGGGCCTCTGCCTCCCAAGTGCTGGGACTTAAGGCATGCTGCATCACCACTCGCCTACTCATTTACACAGAGCCATTCTGTACAGAAGCTCTGGATTCTGCTACGGATGCTGCACTTCCGTTTGCTTGCTTGCTTGTGCTGCTAGAGATGGAACTCGGGCTCATTTCATGCTAAGCAGGTGCTctgtggctccagcctgaagcctcgctttggaaaaattttaatgatattaaaacatgagggttggatgtactctgttcagatttaactcccttttgtgtgtttatcagaatcaagatcattatgcggttcttggacttggccacgtcagatacacagctacacagagacagatcaaagcagctcgtaagtacttacttagtatgggaggtataactccaaactcgggggtgcagcaccctgaagttcatgagaccccatcttagaaagggaagggattagggggaccttgagtgttctcagagttcaagtcctgcaggaagagatcagaaaccctgtaagaatCATTGCCCATCAAGGAAACGGGTTTCGTTTGTTTTGGGTTTGGATGGTGCTGGGAATCAAACCTGAGGCCTTGAGTCCTGGCCAGCTCTGCCTGCCACTGAGCTTCATGCCTAGTCCTCTGCTCTGAGGATTTCTTCCAGGTAAGAGAGAAACAGGGTCTTGCTGTTCAGACCTTAGATCCATGTCCGCCTTCCAAGTGCTGGGATTGTAGACGGTTAGGTATGATCACCATGTTCTCGTCTAATTAAAAAATTAGGTCTCTTTTAGTTTTGCTTCCTAAAAAAGATTTTAATTTTGAAAAATTAGATGTGTGTATCTGTGTGGGTATGTGCACGTGAGTGTAGGTGTGTATCTGTGTGGGT
This mutation is a 3625 bp deletion beginning at Chromosome 19 position 41,924,589 bp and ending after 41,928,213 bp (GRCm38a 342 bp deletion beginning at Chromosome 5 position 21,781,162 bp and ending after 21,781,503 bp (GRCm38/mm10). mm10).
JAX Protocol
Protocol Primers
| Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
|---|---|---|---|---|---|---|
| 48298 | GGG ACC TTG AGT GTT CTC AG | Wild type Forward | A | |||
| 48299 | AAC CCA AAA CAA ACG AAA CC | Common | A | |||
| 48300 | TGC TTG CTT GTG CTG CTA GA | Mutant Forward | A | |||
| 48301 | Fluorophore-1 | AGT CCT GCA GGA AGA GAT CAG A | Quencher-1 | WT Probe | ||
| 48302 | Fluorophore-2 | CTA AGC AGG TGC TCA TTG CC | Quencher-2 | MUT Probe |
Reaction A
| Component | Final Concentration |
|---|---|
| Kapa Probe Fast QPCR | 1.00 X |
| ddH2O | |
| 48298 | 0.40 uM |
| 48299 | 0.40 uM |
| 48300 | 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.