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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= 100 bp
Wild Type = 108 bp
>chr2:101629136+101629243 108bp AAGCTGCTGCCACAATAAAG AAGGCTTGTTTAAAAGAAAAGTCA
Sequence
Wt Sequence (with deletion in lower case):
TCCCAGAAATCAAAGCAAAGCCAGTCCGCCACTAAAATAGGTCCAAGCTGCTGCCACAATAAAGTAGTGTCTGTGATTtcctaacgggggcaggaggtgtcttataattctaagacaagcatgtaaaatgacttttcttttaaacaagccttttgtatgaggaagaatctaagcataattaccaatatgaaaagatattctaagttattgcgtttttttaatcctttcagataaaagacctattcacaatcaaaaatgtccctgcagatggtaacagtgggtcataacatagccttaattcaaccaggcttctcacttatgaattttgatggccaagttttcttctttggccagaaaggctggcctaagagatcctgtcctactggagtct…………..aataccaataaataaactaatgcacaaaaagttaagttcttttgtgtaataagtagcctatagttggtttaaacagttaaaaccaacagctatatcccacactactgctgtttataaattttaaggtggcctctggtttatacttatgagcagaattatatatattggtcaataccatgaagaaaaatttaattctatatcaagccaggcatggtgatggtgatacatgcctgtaatcctggcacttaggaagtggaagaaggaagtttgtgagtttgatgcttgttgaggtatgaccttttgctatgtattgtagtgtatgagccccaagacctgcttgacccagagacaagagagtccacacatagatccaagtaatgctatgtgaccttgccccccggttacttgtgattAGGTGAATAAAGATGTCAACAGCCAATAGCTGGGCAGAAGAGCCAAAAGTGGGGATTGAGGGTACCCTGGCTTGAT
JAX Protocol
Protocol Primers
| Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
|---|---|---|---|---|---|---|
| 48593 | AAG CTG CTG CCA CAA TAA AG | Common | A | |||
| 49187 | AAG GCT TGT TTA AAA GAA AAG TCA | Wild type Reverse | A | |||
| 49188 | Fluorophore-1 | TTC CTA ACG GGG GCA G | Quencher-1 | WT Probe | ||
| 49189 | GGT ACC CTC AAT CCC CAC TT | Mutant Reverse | A | |||
| 49190 | Fluorophore-2 | AGG TGA ATA AAG ATG TCA ACA GCC | Quencher-2 | MUT Probe |
Reaction A
| Component | Final Concentration |
|---|---|
| Kapa Probe Fast QPCR | 1.00 X |
| ddH2O | |
| 48593 | 0.40 uM |
| 49187 | 0.40 uM |
| 49189 | 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.