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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
>chr18:12685013+12685169 157bp GAGATAAGTGTTCTCCCCCAAG CCAAGACCTTTCTTCTTGTCA
Mutant= 138 bp
Wild Type = 157 bp
Sequence
Wt Sequence (deletions in lower case):
TAGATGTTTTCTGTTTTGTTTCGACCCAGGATCTTGCTTATGGCTAGCTTGGGTAAACTCCGCCGTGGTCCTCCAGCTTCAGCCTTCCAAGCACTGCAGGCCTGCACCACCATGGCCACCCCTTATTGTAAAGCTTGGATTCAGCTTTGTGCTTAGCAGCAGATCAGCACCttcctttggccatgatctagcaaatctttctatttatatctttatatttactgtcagctccttagtgttataattttacactgagacctcccttgcttttgaagaatcaaagatggagtctctcttggtcgcagagccaggaagtacctcagctcttcaacttcttggtgtaatttactagtgaatggggtgcggtctatgtaatgatatatcctaattaccaaatgcttttatagtaagacttctagtgaaatatttttgtttgttctttaacagaaatcatagtccactaatgagttttggagccagcttcgtcagctttttggtaagttgatatcaaaagagatattgctgtcaagaaaataaattcaaagtgatgcctatttactaccagctgtatagacatactgttaaagaaaacttaaagttattacccattatttaatgtctgtgagataagtgttctcccccaagtataaaaaaaaatgcgtatatatgatttttagagggTTATATTTTCCCCAGCTTGAGATATGTTTTTAGAATAAAAATCTAATTGAAAGCTCTTCAAAGCTTAAGATTGTATTGTGACAAGAAGAAAGGTCTTGGTAAGGAAGTTAACAACAGCCTAGAGATGAGTTTGTGAGCTAAACTAAAGCAGTTTAATACTTGAGAGTGGAAGACACGAGTTACAGTAGTGTTATTCAGTCAGCTGGTCAATATA
This mutation is a 510 bp deletion beginning at Chromosome 18 position 12,817,618 bp and ending after 12,818,127 bp (GRCm38/mm10).
JAX Protocol
Protocol Primers
| Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
|---|---|---|---|---|---|---|
| 64648 | GAG ATA AGT GTT CTC CCC CAA G | Wild type Forward | A | |||
| 64649 | CCA AGA CCT TTC TTC TTG TCA | Common | A | |||
| 64650 | GCT TGG ATT CAG CTT TGT GC | Mutant Forward | A | |||
| 64651 | Fluorophore-1 | ATG CGT ATA TAT GAT TTT TAG AGG GT | Quencher-1 | WT Probe | ||
| 64652 | Fluorophore-2 | AGC AGC AGA TCA GCA CCT TAT A | Quencher-2 | MUT Probe |
Reaction A
| Component | Final Concentration |
|---|---|
| Kapa Probe Fast QPCR | 1.00 X |
| ddH2O | |
| 64648 | 0.40 uM |
| 64649 | 0.40 uM |
| 64650 | 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.