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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
>chr5:53699686-53699805 120bp TGTCCCGGTATTCCTACAGC CCTCCCACTTCCTCTTCCTC
Mut= 89 bp
Wt= 120 bp
Fam=Mut
Hex=Wt
Sequence
Wt Sequence:
GAACAAACGCTTTCGCCTGGGCTTCATGGCCACCTTCCCCTGTTGCCCGAATCCTGGTCCCACCGGGGTGAGAGGAGAAGTGGGAGAGGAGGAAGATGGAAGGACCATAAGGGCATCcctgtcccggtattcctacagccacatGAGCACCTCTGCCCCACCCCACTGAGCTGTACCTGGTTCGCTGCGGGCAGCAGAAAGGAGGGGACTGGGAGCAAGGAGGAAGAGGAAGTGGGAGGGAAGGAGAAGGAAGACCCATTTCTAACGAGGACTCTTCCGATGTCTGCTTTTCACCCTTCACAAAGTTCCAGAAGGAACTTAAGGCGGGGTCATGAAAACGCTAAGACATTTTC
Mutant Sequence:
TTCGCCTGGGCTTCATGGCCACCTTCCCCTGTTGCCCGAATCCTGGTCCCACCGGGGTGAGAGGAGAAGTGGGAGAGGAGGAAGATGGAAGGACCATAAGGGCATCACTGTCCCGGTATTCCTACAGCCACATGAGCACCTCTGCCCCACGAGGGCAGAGGAAGTCTTCTAACATGCGGTGACGTGGAGGAGAATCCCGGCCCTATGGGGCCCAAGAAGAAGAGGAAAGTCTCCAACCTGCTGACTGTGCACCAAAACCTGCCTGCCCTCCCTGTGGATGCCACCTCTGATGAAGTCAGGAAGAACCTGATGGACATGTTCAGGGACAGGCAGGCCTTCTCTGAACACACCTGGAAGATGCTCCTGTCTGTGTGCAGATCCTGGGCTGCCTGGTGCAAGCTGAACAACAGG
JAX Protocol
Protocol Primers
| Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
|---|---|---|---|---|---|---|
| 63007 | GGA TTC TCC TCC ACG TCA CC | Mutant Reverse | A | |||
| 64160 | TGT CCC GGT ATT CCT ACA GC | Common | A | |||
| 64161 | CCT CCC ACT TCC TCT TCC TC | Wild type Reverse | A | |||
| 64162 | Fluorophore-1 | AGC TGT ACC TGG TTC GCT G | Quencher-1 | WT Probe | ||
| 64163 | Fluorophore-2 | AGG GCA GAG GAA GTC TTC TAA CAT | Quencher-2 | MUT Probe |
Reaction A
| Component | Final Concentration |
|---|---|
| Kapa Probe Fast QPCR | 1.00 X |
| ddH2O | |
| 63007 | 0.40 uM |
| 64160 | 0.40 uM |
| 64161 | 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.