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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
>chr1:104940912+104941051 140bp CTGAAGGGGAAAGAACATTC CTGTCCATGTCTGAATGAAGC
Mut= 150 bp
Wt= 140 bp
Fam=Mut
Hex=Wt
Sequence
Wt Sequence (deletion in lower case):
ACAATAATTTTAAAATATTAAGATCATACTGTGTTTGAATACCATAAGAATATGACATAGGAAAACTTCACATATTATTCTTTATCCATTGGTTCTTTACAGACAGACATCCTAAACACTGAAGGGGAAAGAACATTCTTATGTAACGGGAGATCTTAAGTCTGTTCCAAAGTGAAAATCAACTTATTTCCAatacctgtgcatatcaataatgacatttcagtgcttttgtccccagcttcattcagacatggacagaggagatggatccatcaaatacatcctttcaggagaaggagcaggcatcgtgtttaccattgatgacaccactggagacatccatgccattcagaggctcgaccgagaagaaagagcccaatatactctaagggctcaagccctagacaggagaacaggccggccaatggagccagaatcagagttcattatcaaaatccaagatatcaatgacaacgagcccaagttcctggatggaccttacatcgccactgtgccagaaatgtcaccagtaggtaagatggtggtgagctcatttttacaaatagtagatgcattgtaggagatgctactttgtacacatagaggagaaaaggtacagaaatcataaaagtgaaactaaaatgactaccaactccttcaataggtagcttcctgaagctatggagatatcagaagcagccacattaagtaataattttccaatgcctgttagttctgagttcttaagattcaaagacatgaacaatggctgatagattatattgtattagattagattagattagattagattagattagattgttaaggtaaatattggtattaccaataaacaaggaccacataggcagtgttacttttactggctactaagaatttaacacagctgggcgtggtggcgcatgcctttaatcccagcacttgggaggcagaggcaggtgaatttctgagtttgagggcagcctggtctacagagtgagttccaggacagccagggctacacagagaaaccctgccttgaaaaataaaataaaataaaataaaataaaataaaataaaataaaataaaataaaatttaaaaaaaagaaagaaagaatttaacacatatgactatcatccctattgagtaacaaaatgggagtcatggaatcgtgatcaagaaagggaactctcccagagaagcaagcaacatctgtaaAATGTTTTAAGGTTGACTATGCAAGTACTATACCAGCTGTTTTAAACTGGGCTTCATTAGGTTTCTCCCACTTCTCTCCCTCAGGTACATCTGTTATCCAAGTGACAGCCACAGATGCTGATGACCCAACCTATGGCAACAGTGCCCGGGTAGTGTACAGCATTCTCCAGGGCCAGCCATATTTTTCTGTGGACTCCAAAAC
This mutation is a 1028 bp deletion beginning at Chromosome 1 position 104,940,986 bp and ending after 104,942,013 bp (GRCm38/mm10).
JAX Protocol
Protocol Primers
| Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
|---|---|---|---|---|---|---|
| 53808 | CTG AAG GGG AAA GAA CAT TC | Common | A | |||
| 53809 | CTG TCC ATG TCT GAA TGA AGC | Wild type Reverse | A | |||
| 53810 | GAG AAG TGG GAG AAA CCT AAT GA | Mutant Reverse | A | |||
| 53811 | Fluorophore-1 | ATT TCA GTG CTT TTG TCC CCA | Quencher-1 | WT Probe | ||
| 53812 | Fluorophore-2 | ACT ATA CCA GCT GTT TTA AAC TGG GC | Quencher-2 | MUT Probe |
Reaction A
| Component | Final Concentration |
|---|---|
| Kapa Probe Fast QPCR | 1.00 X |
| ddH2O | |
| 53808 | 0.40 uM |
| 53809 | 0.40 uM |
| 53810 | 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.