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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
>chr16:8664144-8664268 125bp AGGACCTTCTCAGCGTAAGTCT GCACCCTCCTGTTTTCTCAC
Mutant= 97bp
Wild Type = 125bp
Sequence
Wt Sequence (deletion in UPPERCASE; insertions with carrots ^g^ (g insertion):
ttatacatgggttcaaacacaggttctcctgcttgtgtggcaactattttactgactagaccatcaccctagcacataacctggcatttcctttcccagtggagatggagggaggctggtaggtgccatgggagcccagggctggcacctgagcagacacttgagccaaggtgtgactatagacaaggtgatctggagtgagc^tcactcaca^CTCTAGGAGTCTCAAGCTCTGTCCTGTCTAGTCTTCTGAGGCAGGACTCTGAGAGCCCCCTTCTCTCTCTAGGTCAGCCATGTCATCTGAGCCTCCTCCACCGCCGCTGCAGCCCCCCACGCATCAGACTTCTGTAGGGCTATTGGATACCCCTCGCACCCGGGATCGCTCTCCATCCCCCCTGCGGGGCAATGTGGTCCCTAGCCCTCTGCCTACCCGCCGGACAAGGACCTTCTCAGCGTAAGTCTTCGGCTGGTCAAGCACTGTTCCCCAAATGGGGCTGCTACCAGATCTAGCAGGATCTACTCCTAGAGACTAGCTAGAAGTACTAGTGAGAAAACAGGAGGGTGCCTTTAAAACTCAGGGTTAAAGGCCATCCTTGCCACCACCCGTGGGAACTGGCCCCTGACTGTAGTGTTAAGTTTTTGGACTTTAGTCAAAGAAGCCTTTTTCTATGAGGCCGTGGGGGAGAGGGATGGGACGAGATACAGGACAATGAGTGTCAGGGTAGCTACTGGCAGGTGGAGAATTTAGGGCCGAGAGGTGTTGCTAAGTTAGCTCAGTTCCCTGTTAACAAATGGACAAACAGGCCTGAGAAAAACACGCCACGGTTGCCCAGTCATTTCTACTGTGTGAATGGCTCCCAGGGCTGAACACAAAGCCTGTGTGTGACTGTTCATGGTGGCCCCCAGCCAGGCCTTCCTGGGACAACTGGGAAAGTAGCTCCAGGGGAGTGGTGGCTCTTGTAGAGGACCGACGGCTGTCTCCTTGCATTTCCCTCTCAGGACAGTGCGGGCTTCGCAGGGCCCCGTCTACAAAGGAGTCTGTAAATGCTTCTGCCGGTCCAAGGGCCACGGCTTCATCACCCCAGCTGATGGTGGTCCTGACATCTTCCTGCACATCTCTGAGTGAGTTAACTGCTGCATATCTGGAGGTTTGGGGAGGCTTGGGGTGGGAGCAGAGGGGTACTGTTGTACCACTGTAATGGACGGTGCTGGGCAGGAGAGCATCTATGGGGTGCGTGAGGCTGGGCAGAGCTCCGTGGGACAGAGACTGGGCATGTCTAGGAATTGAGTAATGTCTTGGGTGTATGTCCACCACAGATGGTaggaacaagatgaggacagttgtgcctggcatgtctaactgggtctgagtgtccagtgcctgacctgtggagagggtgtccctgggatggggatatgtgttggccttcttaaaggaggggcctggtgaggctggggtctgttccttttgctccaggaaggaagctcagggctgggctctttctcctgccac
This mutation is in a 1108 bp deletion beginning at Chromosome 16 position 8,663,387 bp and ending after 8,664,494 bp (GRCm38/mm10).There is a 9 bp insertion (TCACTCACA)at the deletion site.
JAX Protocol
Protocol Primers
| Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
|---|---|---|---|---|---|---|
| 67739 | AGG ACC TTC TCA GCG TAA GTC T | Wild type Forward | A | |||
| 67740 | GCA CCC TCC TGT TTT CTC AC | Wild type Reverse | A | |||
| 67741 | GCA CCT GAG CAG ACA CTT GA | Mutant Forward | A | |||
| 67742 | CCA GGC ACA ACT GTC CTC AT | Mutant Reverse | A | |||
| 67743 | Fluorophore-1 | AGC AGG ATC TAC TCC TAG AGA CTA GCT | Quencher-1 | WT Probe | ||
| 67744 | Fluorophore-2 | TGG AGT GAG CTC ACT CAC AAG GAA C | Quencher-2 | MUT Probe |
Reaction A
| Component | Final Concentration |
|---|---|
| Kapa Probe Fast QPCR | 1.00 X |
| ddH2O | |
| 67739 | 0.40 uM |
| 67740 | 0.40 uM |
| 67741 | 0.40 uM |
| 67742 | 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.