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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
>chr3:89651898+89651981 84bp CTCAAAGTCTTATACCATTCCCTTG CACACAGTTAGGAGGTAAAACACA
Mut= 114 bp
Wt= 84 bp
Fam=Mut
Hex=Wt
Sequence
Wt Sequence:
acaagctttatggcatgtcttgaccaattagagactcaaagtcttataccattcccttgaaatgccaacactaagcctggggcacaccaagtcttgtgttttacctcctaactgtgtgttttctctcctgggaccctctgacgtcctctggcagGGTGCAGGCTGCAC
Mutant Sequence:
GGGCGCAAGCAGGTTTTGACAAGCTTTATGGCATGTCTTGACCAATTAGAGACTCAAAGTCTTATACCATTCCCTTGAAATGCCAACACTAAGCCTATAACTTCGTATAGCATACATTATACGAAGTTAtCACACCAAGTCTTGTGTTTTACCTCCTAACTGTGTGTTTTCTCTCCTGGGACCCTCTGACGTCCTCTGGCAGGGTGCAGGCTGCACTGCCCTTGTGG
JAX Protocol
Protocol Primers
| Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
|---|---|---|---|---|---|---|
| 56863 | CTC AAA GTC TTA TAC CAT TCC CTT G | Forward | A | |||
| 56864 | CAC ACA GTT AGG AGG TAA AAC ACA | Reverse | A | |||
| 56865 | Fluorophore-1 | AAG CCT GGG GCA CAC C | Quencher-1 | WT Probe | ||
| 56866 | Fluorophore-2 | CAC TAA GCC TAT AAC TTC GTA TAG CAT | Quencher-2 | MUT Probe |
Reaction A
| Component | Final Concentration |
|---|---|
| Kapa Probe Fast QPCR | 1.00 X |
| ddH2O | |
| 56863 | 0.40 uM |
| 56864 | 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.