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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
Mutant= 141 bp
Wild Type = 154 bp
>chr7:64578668-64578821 154bp GATCAAAAACTGTTCCCATTCT GCACTGTCCATGTGGAATGA
Sequence
Wt Sequence: GTGTATGTCTCCTTAAATGAAAATGGTGACAAGAAGCCATGTTTTCATTTTTTTCTTTGGAGTTGGGAGCCACTAGAACTTTGCTTTGAAACCGTAGGCTTTTTATCTACCACTGATATTGCTCATCCCATGATTTAGCTTTCTAGCAAGTTTGACATTAAATTATTTGTGCAGTGTCTCATATTTTATCTCATTTCTTTGCCATCAAAATCAACTTAAGTAACATCGAAAATCtttatagtcatctgttactggcaatttgcagtggggtagctgtcttgtttcccaaaagctcaataaatacgagtcaggatctctcatcctttgaagttttaagaagacaagatgaatttagttttaaatgcaaattaagtccccaagagaaatctgcccacctgagcagcatgggcctgtgttgttagtggctgaggcttctagacactgattggtttgtgctcttcatgtttcactgtaccataaagtactaacagatatgtatttttcccccacagtaaaaccaactgtgatcgatgtggacatttatgttaacagcattggtcctgtgtcatccataaacatggtaagaaacacctgtgtttaccttggtagctcctgtgcgtcttcatgcttacagtctccactggatttctgttgcagttttattaagcagatttaaagcccacgactgactcaagtgttaagtgccgtggtattgaaaatagtctttaagaatcagaagggacagtgtgacatcagcagtgagtcattgctctgaagatcaaaaactgttcccattcttacatacatagatagcacatgccattcaaagggaattatttagggttgtgtactcatTGGCTTTAGGCAACCTCACTAATATTAGGGATGCACGTAAGTCCAGTGAGAGAAGTCATTCCACATGGACAGTGCTTAGCTGCTAAGCACGCGATGCTGTACAAGAATGCATTACACCAAGCCACCTGACATGTCTGCCAGCTGTGGTGTGCCTCCTGATTAGATGATGTAGAAGTCCCATAGCAGGTCATT
Mutant Sequence:tttatagtcatctgttactggcaatttgcagtggggtagctgtcttgtttcccaaaagctcaataaatacgagtcaggatctctcatcctttgaagttttaagaagacaagatgaatttagttttaaatgcaaattaagtccccaagagaaatctgcccacctgagcagcatgggcctgtgttgttagtggctgaggcttctagacactgattggtttgtgctcttcatgtttcactgtaccataaagtactaacagatatgtatttttcccccacagtaaaaccaactgtgatcgatgtggacatttatgttaacagcattggtcctgtgtcatccataaacatggtaagaaacacctgtgtttaccttggtagctcctgtgcgtcttcatgcttacagtctccactggatttctgttgcagttttattaagcagatttaaagcccacgactgactcaagtgttaagtgccgtggtattgaaaatagtctttaagaatcagaagggacagtgtgacatcagcagtgagtcattgctctgaagatcaaaaactgttcccattcttacatacatagatagcacatgccattcaaagggaattatttagggttgtgtactcat
JAX Protocol
Protocol Primers
| Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
|---|---|---|---|---|---|---|
| 37071 | GCA CTG TCC ATG TGG AAT GA | Mutant Reverse | A | |||
| 37072 | GTG CAG TGT CTC ATA TTT TAT CTC A | Mutant Forward | A | |||
| 37073 | TCC CCC ACA GTA AAA CCA AC | Wild type Forward | A | |||
| 37074 | CAG TGG AGA CTG TAA GCA TGA AGA | Wild type Reverse | A | |||
| 37075 | Fluorophore-1 | AGG GAT GCA CGT AAG TCC AGT GA | Quencher-1 | MUT Probe | ||
| 37076 | Fluorophore-2 | TGG TCC TGT GTC ATC CAT AAA CAT G | Quencher-2 | WT Probe |
Reaction A
| Component | Final Concentration |
|---|---|
| Kapa Probe Fast QPCR | 1.00 X |
| ddH2O | |
| 37071 | 0.40 uM |
| 37072 | 0.40 uM |
| 37073 | 0.40 uM |
| 37074 | 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.