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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= 104 bp
Wild Type = 70 bp
>chr7:79461430-79461499 70bp TTGAAACTGTTGTGGCAGATG CAACACAGCAGGGATGGAG
Sequence
Wt Sequence:
AGTGGGCCCACACACCTTCCTGCCCATGGCACCCAGTCATAGCCGTTAGCTCTGTTAATCTGTGGGAGGCGAATAGTAAAGGGGCTGTGTCATTTCTCTCCTCAATGATACCCTGACTTCTGTGACAGTGAGTGGCGTGAGGAGCAGCCCTTGGGGACCTGGCATGTGTGGGTGGTGTGGAATAGAAGTCTAGTCCTTCGTGGTAACCCTGTCCACTCTTGCCTCTTTCAGGTATTCCTGGTGCAGCCGGCGGCTGGTGGAAGAGCGTTACTCTTGGACCAGCCAGCTATCGCCGGCTGACCTAATCCCTTTGGGGGGCTCCACTAGTGCCAGCAGCTCCACCAAGCAGGATGGGCAGGAACAGTTAGTGGTGGGGCACAATGTTTCCTTTGACCGAGCCCATATCAGGGAACAGTATCTGATTCAGGTAAGGTTTGTGGGAATGGAGTGCTGTAACATAAGGTGGGCCAAAAGCCCTGTCACCTTTGCTAGGAAAAGCCTCGCTTTCTCCGTGACTGTTTCCACCCCCTTTCCAGCATTGCTGGGACGTCTGTTGACCAGCTTTGCTTGATCTCTGCTCAGCTGCTGATCTCCAGAACAGACTTACAACACACTAGCATGGTGTTTGAAACTGTTGTGGCAGATGACTCCTGTCTTCCCTAAAGACCGCAGGGAGCTCCATCCCTGCTGTGTTGGCGCAGGACTGGGAGTTAGTCCAGGATAAAATGGCTCTTATCTTAGAATGGCATTTTAGAGTTTTTGAGGGCTCTGAGTTACAGCCTGGGCCACATCCTGCCTCAGCTGTTGATGGGCAGTATTTTTTCCTTAACATCTCTAAGCCCCAGTCTCTATAAAGAGAGATAACAGAAGCTCCCTCAGTGGACTTTGAGCAGAGTTTGAATAGCACACATGT
Mutant Sequence:
AAGGTCTCAGAGCTTGCTGCATACCCAGTGGGCCCACACACCTTCCTGCCCATGGCACCCAGTCATAGCCGTTAGCTCTGTTAATCTGTGGGAGGataacttcgtatagcatacattatacgaagttatCGAATAGTAAAGGGGCTGTGTCATTTCTCTCCTCAATGATACCCTGACTTCTGTGACAGTGAGTGGCGTGAGGAGCAGCCCTTGGGGACCTGGCATGTGTGGGTGGTGTGGAATAGAAGTCTAGTCCTTCGTGGTAACCCTGTCCACTCTTGCCTCTTTCAGGTATTCCTGGTGCAGCCGGCGGCTGGTGGAAGAGCGTTACTCTTGGACCAGCCAGCTATCGCCGGCTGACCTAATCCCTTTGGGGGGCTCCACTAGTGCCAGCAGCTCCACCAAGCAGGATGGGCAGGAACAGTTAGTGGTGGGGCACAATGTTTCCTTTGACCGAGCCCATATCAGGGAACAGTATCTGATTCAGGTAAGGTTTGTGGGAATGGAGTGCTGTAACATAAGGTGGGCCAAAAGCCCTGTCACCTTTGCTAGGAAAAGCCTCGCTTTCTCCGTGACTGTTTCCACCCCCTTTCCAGCATTGCTGGGACGTCTGTTGACCAGCTTTGCTTGATCTCTGCTCAGCTGCTGATCTCCAGAACAGACTTACAACACACTAGCATGGTGTTTGAAACTGTTGTGGCAGATGACTCCTGTCTTCCCTataacttcgtatagcatacattatacgaagttatAAAGACCGCAGGGAGCTCCATCCCTGCTGTGTTGGCGCAGGACTGGGAGTTAGTCCAGGATAAAATGGCTCTTATCTTAGAATGGCATTTTAGAGTTTTTGAGGGCTCTGAGTTACAGCCTGGGCCACATCCTGCCTCAGCTGTTGATGGGCAGTATTTTTTCCTTAACATCTCTAAGCCCCAGTCTCTATAAAGAGAGATAACAGAAGCTCCCTCAGTGGACTTTGAGCAGAGTTTGAATAGCACACATGTCAGACAACCAGAGCTGTCTGATGTGTGGTAGTGACAGTTCTTAGATTATAAAAAACCCTTTTTCTTTGACTCTTCACAGCAGGGTAAGGTGAATGGCAGG
JAX Protocol
Protocol Primers
| Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
|---|---|---|---|---|---|---|
| 59439 | Fluorophore-1 | TG+ TA+ T+G +CT ATA +C+ GAA G | Quencher-1 | MUT Probe | ||
| 63361 | TTG AAA CTG TTG TGG CAG ATG | Forward | A | |||
| 63362 | CAA CAC AGC AGG GAT GGA G | Reverse | A | |||
| 63363 | Fluorophore-2 | TCT TCC CTA AAG ACC GCA G | Quencher-2 | WT Probe |
Reaction A
| Component | Final Concentration |
|---|---|
| Kapa Probe Fast QPCR | 1.00 X |
| ddH2O | |
| 63361 | 0.40 uM |
| 63362 | 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.