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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 transgene genotype is determined by comparing ΔCt values of each unknown sample against known homozygous and hemizygous controls, using appropriate endogenous references.
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
>chr12:69959693+69959786 94bp ACCGTGTGAGAAGTTAATAGTGTTAG CAGTGCTAGTCAAGGGCTAAGT
Mutant= 116 bp
Wild Type = 94 bp
Fam=Mut
Hex=Wt
Sequence
Wt Sequence:
atttattatgaaaattctgcactgcatgaattgtatacgttatactttaagcaatagcagttgaataggctgttcatagtaactgtgctctgactgacaacacagttctggctgcagtgtctcgaggtggtggaagtgtagcccaccccttagtccatcactcttaggaagcattctggtgtactccaagaaaaggcatggacacagttgttaatttactccatattacataaacagaagcctctctttctgacagGTTTGTGGTGGTGATAAACCATTTTTGGCCCCTAACGACCTGCAAAGCAAGCACCTGCAGCTAAAAGAAGAATCTGTGAAGTTATTCCGAGGCGTGAAGAAGATGGGCGGGGAAGAGTTCAGCCGGCGTTACCTGCAGCAGCTGGAGAGTGAAATAGATGAACTCTACATCCAGTATATTAAGCACAATGACAGCAAAAATATCTTCCATGCTGCTCGAACCCCAGCCACACTGTTCGTTGTCATCTTTATCACATACGTGATTGCTGGTGTGACTGGATTCATTGGTTTGGACATCATAGCTAGCCTGTGCAACATGATCATGGGACTGACGCTTATCACCCTCTGCACCTGGGCATATATCCGCTACTCTGGCGAATATCGAGAGCTGGGTGCTGTAATCGACCAGGTAGCTGCAGCTTTGTGGGACCAGgtgagagtaccttcaggtttgcacatgctgtgtaattagtgttgcattcactattagaaatatacaggtgaacatgacttgggcccattctttaaaactttacagtataccaagatttgagcaactaactaagtcctacacaactgctttctagttaacattttagtatttaaaacatacatacattaacagcatttttactctctgattctaaaatattaccgtgtgagaagttaatagtgttagtagtgtctgacagtcatttagctgtcgtgaccttgagttaaacttaacttagcccttgactagcactggcttggtgaggggcatatgtgaaccacaatcatttggttatatgtcattccctttaagtatcagattacttcagactg
Mutant Sequence:
ATTCTGCACTGCATGAATTGTATACGTTATACTTTAAGCAATAGCAGTTGAATAGGCTGTTCATAGTAACTGTGCTCTGACTGACAACACAGTTCTGGCTGCAGTGTCTCGTCCATGTCAAAAAGAGAAAAATTTAAAATGATTTTAAGCCACCTATAAAATACCAGTCCCATTTATAGTCATGCCTCGTGGATAGGGGGTGGAAAGATGTGGGCTGACAAAACATATATGCAGGCTCCTGATTATTAACGTATTCTAACAAACTCAACTAGCTAAGTGAAAAATTTTGATACAGTTGCCAATTTTACTCTGCATTGCATAAACAAATACTTCTCTATCTGATACAGATTTGTGGTGGTGACAAACCATTTCTGGCCCCAAATGACTTGCAGACCAAACACCTGCAACTTAAGGAAGAATCTGTGAAGCTATTCCGAGGGGTGAAGAAGAtGGGTGGGGAAGAATTTAGCCGGCGTTACCTGCAGCAGTTGGAGAGTGAAATAGATGAACTTTACATCCAATATATCAAGCACAATGATAGCAAAAATATCTTCCATGCAGCTCGTACCCCAGCCACACTGTTTGTAGTCATCTTTATCACATATGTGATTGCTGGTGTGACTGGATTCATTGGTTTGGACATCATAGCTAGCCTATGCAATATGATAATGGGACTGACCCTTATCACCCTGTGCACTTGGGCATATATCCGGTACTCTGGAGAATACCGAGAGCTGGGAGCTGTAATAGACCAGGTGGCTGCAGCTCTGTGGGACCAGGTAAGAACACCTTTAATTCACAACTAAATTCAGCACACATTTGAATGTCATCCATGTGCCAGCCACTGCATTAGATGTTGGAAATACAGATCAACAGGAATTGGGCCCCAGTCATCAAGAATGTTATGACCTGCCCAGATACAAGCAACTAACTATATAATACTGTGTAAAATGTGAAGTCCTACACAACTATTTTCCAGTTAACATTTCAGCATTTAAAAAAATATACATCAGTTTAGTAGTATATATACGCTTGGTGAGGGGCATATGTGAACCACAATCATTTGGTTATATGTCATTCCCTTTAAGTATCAGATTACTTCAGACTGTAATTGACCTATACTTCTGCACTAAGCTGCAG
JAX Protocol
Protocol Primers
| Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
|---|---|---|---|---|---|---|
| 59846 | CAG ACC AAA CAC CTG CAA CT | Mutant Forward | B | |||
| 59847 | TCT ATT TCA CTC TCC AAC TGC TG | Mutant Reverse | B | |||
| 60639 | ACC GTG TGA GAA GTT AAT AGT GTT AG | Wild type Forward | A | |||
| 60640 | CAG TGC TAG TCA AGG GCT AAG T | Wild type Reverse | A | |||
| 60641 | Fluorophore-1 | ACA GTC ATT TAG CTG TCG TGA CC | Quencher-1 | WT Probe | ||
| 71539 | Fluorophore-2 | TGA AGA AGA TGG GTG GG | Quencher-2 | MUT Probe |
Reaction A
| Component | Final Concentration |
|---|---|
| Kapa Probe Fast QPCR | 1.00 X |
| ddH2O | |
| 60639 | 0.40 uM |
| 60640 | 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.
Reaction B
| Component | Final Concentration |
|---|---|
| Kapa Probe Fast QPCR | 1.00 X |
| ddH2O | |
| 59846 | 0.40 uM |
| 59847 | 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.