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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= 99 bp
Wild Type = 116 bp
>chr6:128366298-128366413 116bp ATGGAGGTCACATGACAAGC CGACCCCAACACAAGTCAAC
Sequence
Wt Sequence (deleted region in lower case)
CCTGGTTGGCCTGGAATTCACACCAGTCCTTCTGCCTCGGCCTTATGAGTGCTAGAGGGGTATGGGTGTGCACCATACATTGGAAAAAATTTTCTTTTTTAATTTCTTTCCCTAAATCTGAGATTTTTACCTCCTTAAGTATCCACTTTgCTATATGGACAAACCAAACTGTACAGCTCAGTGTGTCTGTTTATTAATAAAGACAATTTATGGAGGTCACATGACAAGCTTTCATGAATGCTTTGGAAGTTTCGAGATgaatgttctctcttggtacctctctcaggttccctttctctcttgcagttgacttgtgttggggtcggagatgtctgtaataaagggaaggtaagcacgtgggtgagggtagagctccaggcccttgcctccattgtgactcccagggactcccagggaacgtcagcctctgataagctctccccaaccctcgtgtctctcctgtgcctctctgtctgtctctgtctgtctgtctgtctgtctgtctccctctctctctttctctctctctctctctcccagaacctggtggtagcagtcagcgcagaaggctggctgcacttgtttgacctgacacccaccaaggctctggatgcctctgggcaccatgagacacttggagaggagcagcgacctgtcttcaagcagcacatcccggccaacacaaaggtcatgctgatcagcgacatcggtgggcatgagcagccaaggggcgggggccagtgggttcctgggcagaccccagggGACTGGGCCAAGATGGAGAGTTGTTGCCATAGCAACCCTAGGTGTGGAGAGCTGGCACCTTGgtGCACATGTGCAGTGTTTACCTTCTCTCTTTGAATTTTGTTTTACTTTCTCCTCATTTGTTGTATGTTTAATGTATAGCTACTGAGCTCCTGTTACAAGCCAGGAACTGTTCTAGGTTCTAGAATTACAAAAATGAATGAAGCAAGCAAAAAAAAAAAAAAAAATCATGACCTCTAAGAATAGACAGAA
507 bp deletion beginning at Chromosome 6 negative strand position 128,416,346 bp and ending after 128,415,840 bp (GRCm38/mm10). In addition, there are 2 small intronic indels, a 1 bp (G) deletion 108 bp before the exon deletion and a 2 bp deletion (GT) 62 bp after the deletion
JAX Protocol
Protocol Primers
| Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
|---|---|---|---|---|---|---|
| 38329 | ATG GAG GTC ACA TGA CAA GC | Common | A | |||
| 38330 | TCT CCA CAC CTA GGG TTG CT | Mutant Reverse | A | |||
| 38331 | CGA CCC CAA CAC AAG TCA AC | Wild type Reverse | A | |||
| 38332 | Fluorophore-1 | TGG GCC AAG ATG GAG AGT T | Quencher-1 | MUT Probe | ||
| 38333 | Fluorophore-2 | TGG TAC CTC TCT CAG GTT CCC | Quencher-2 | WT Probe |
Reaction A
| Component | Final Concentration |
|---|---|
| Kapa Probe Fast QPCR | 1.00 X |
| ddH2O | |
| 38329 | 0.40 uM |
| 38330 | 0.40 uM |
| 38331 | 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.