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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
>chr8:105086275+105086382 108bp TTTGAACAAAGGGTGTTGTCTC TAGCAGGACAGCCAGTCAGA
Mutant= 109 bp
Wild Type = 108 bp
Sequence
AACCAAACATGAATGTTGAATTTGAACAAAGGGTGTTGTCTCTAACTGGGGCTAGAgtcctagcagggaatgaggacaaagagccatcttcaccatctaagagccagctctgactggctgtcctgctacatctacccacaacacatactaggaaaacaggtgattcacgggcaagcaaggactgggaagaggagaagtgcccagtgccctggtgactgactcaaagcaaatgcacagagtgtaagaaaaaaaatatgtcttggacgctatcttaagccaatcagtgccactccctgtccctagcactggagacaagcacatgccaggcaacaggggattcctggatgtggtggctgctcttcgctgggtccaggggaacatagccccctttgggggtgatcccaactgtgtcactatcttcggtaactctgctggaggcatgattgtctcatccctggtgagtgaccacagagaactagcagtcgaggttatatctgggaaggcttctatggcctcagacctccatgctcctctgcctcctaacatctgcccctctgttgttcccagttcctgtctccaatatctgctgggctcttccacagagccatatcccagagtgggattgtcacgaccattatgatggaagacatgaaaccatggcctgaagctcaggtctgcctttccttcactctcttaccctaagtgtgcctccacagcccaccctgtgctaacctttgggtcaggtgtggagaggaaggagtaaccactagggagtcactgataggctcagagacaattggctagtgcagtccaggaggtaggcagtcagacaaggctttctagaggaagccaccctggaagaggttatctctggttcttctctggggttatttttgtagatcccccccattccccaacgGATTCCTATATTTCCAGCTACTCTGTTACCTAAGAGTTCGGTCCCCTTTTGAGGAGTAGCCAGAAGTCACCAAGCAATCCCTAATAGGAACTGTGAGTCAAAAGCTTCATGCATAATAAGACAGGGATGGGGCACAGAAGCAGTAAAGCCATCAGGTAGGGAACAAGCAGGAAGTGAATGAACTAAAGGAGTGGAAGGTAGTGCAGGGAGCTGACAGACCATGCCTGGGAGGGAGTTTGGCGGGATGCAAAGGGCTAGAAGAAGCAG
An 863 bp deletion beginning at Chromosome 8 position 105,086,311 bp and ending after 105,087,173 bp (GRCm38/mm10).
JAX Protocol
Protocol Primers
| Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
|---|---|---|---|---|---|---|
| 45670 | TTG AAC AAA GGG TGT TGT CTC | Common | A | |||
| 45671 | TAG CAG GAC AGC CAG TCA GA | Wild type Reverse | A | |||
| 45672 | TTG GTG ACT TCT GGC TAC TCC | Mutant Reverse | A | |||
| 45673 | Fluorophore-1 | TGA GGA CAA AGA GCC ATC TTC | Quencher-1 | WT Probe | ||
| 45674 | Fluorophore-2 | CTG GGG CTA GAG ATT CCT ATA TTT C | Quencher-2 | MUT Probe |
Reaction A
| Component | Final Concentration |
|---|---|
| Kapa Probe Fast QPCR | 1.00 X |
| ddH2O | |
| 45670 | 0.40 uM |
| 45671 | 0.40 uM |
| 45672 | 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.