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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= 96 bp
Wild Type = 115 bp
>chr1:167237034-167237148 115bp GACCAGCAAGAAGGGAGATG GACTCCCAACAACGAGAAGC
Sequence
Wt Sequence (deleted region in lower case)
CATTGCATAGCCCTTGCTCCGGCTCTCGCTCCCACTGATGAGGCTGTGTAGGCGCTGAGTGCCCCGTGCTTACGTAGTCACTTTTTGCTCTGTCAGCATACCATCAGTTCCACTTGAAGATGGAGCGATACTTGGCATTCCGTTTTCATTCACCAGCTTGCAGAGAGGAGGCAGGGCCACTCTCCACACCTTACAGCATGGAGCTCTCAGAGCAGCAGGCGCTGAGGAGGAGGAAGGGACCCTGGGCTCCTTAGACCTTCCTTTCAGCAGGCTGGTCTTAGAGGGGTTTTATTTTTCAAGATCCCAAACCCATCCCCACCCCCACctcggcactgtcttaccgttttagttgactgtatattactagagctcagggaggcgccagcctcgagatgtgcacttcctgtgctcccacacacctctgactcctggctgtcgtcttctctcctcacagtcttccgtttgtgctaagatcgtccagcttttggggcagaatgaggtggattaccaccagaagcaggtggtgatcctgagccaggatagcttctaccgagtcctcacctcagagcagaaggccaaagccctcaagggccaattcaactttgatcacccgggtgagttgggagttgagagatgtgtgtgttgaaggtggatcgggtggtggctgtgtccacgaggcttagcttctcccagtctgttcctcctgtccacacataccataggggcctaatgaccagcttgcagggttatcatgtgcttagaggaaagaatgggttttgggatattttgcagagagctgattgaatcgaatgaatgccagccatgtaagcttgggtagagatgggtcagcccccggagcaaacacaccactggcctttctgattgatgtgtagcagcctgtaccatctccacccacccctcaacccccaccctgccccatgtggtctgcttggtgaccagcaagaagggagatgggagtcttaacgtggacccctacgtgaattctgctgcgagcttagatcagcacagtgctcCTTCAGGTCGTCTGGGCTTCTCGTTGTTGGGAGTCAGCCCTGCTTGGCATGGGACTGCAGTTCTTCCTAGTCCCGGTCTACTTTCCACTTAGCTTGAGCCAATCCCAGGGCCCCCAGCTGACCTTCGAGGAGAGATGGGAGGGGCCATGGCAGCACCAAGTGTAGGCTGTCAGCAGGTAACAAACAGCTCAGTTTGCCAGAACTCATTTAACACACCCTGTTTTAAGATCCAGGGAGTGTTTATATTCTGTCACGGGTGGACACTGGCTTTGGAGAGGCAACATACGGGGTGTGAAGTTTGTTCTGATTTTAGGACAGGTTA
709 bp deletion beginning at Chromosome 1 negative strand position 167,237,777 bp and ending after 167,237,069 bp (GRCm38/mm10).
JAX Protocol
Protocol Primers
| Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
|---|---|---|---|---|---|---|
| 38042 | CAG CAG GCT GGT CTT AGA GG | Mutant Forward | A | |||
| 38043 | GAC TCC CAA CAA CGA GAA GC | Common | A | |||
| 38044 | GAC CAG CAA GAA GGG AGA TG | Wild type Forward | A | |||
| 38045 | Fluorophore-1 | AAG ATC CCA AAC CCA TCC C | Quencher-1 | MUT Probe | ||
| 38046 | Fluorophore-2 | ACG TGG ACC CCT ACG TGA | Quencher-2 | WT Probe |
Reaction A
| Component | Final Concentration |
|---|---|
| Kapa Probe Fast QPCR | 1.00 X |
| ddH2O | |
| 38042 | 0.40 uM |
| 38043 | 0.40 uM |
| 38044 | 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.