For in-depth product & services help, ask our
Technical Information Scientists
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= 82 bp
Wild Type = 93 bp
>chr7:29937735-29937827 93bp GTGGCATTCCAGGGTACAAC CACACGAGTGACAAAAACAACC
Sequence
Wt Sequence (deletions in lower case; bp changes in brackets with wt first and insertions with carrots ^g^ (g insertion): ATTTAGAGTCTGGGTAGGTGTGCTGACTTCACTTAAGGAAGGTTCGGGACAGTAGCATCTTAGGCACTGTGATTACCAGTGTGATGGGCAGGATTGAAGCTTCCCAACATGCTGAATTCACCATGACAACAGGGGAGGCCAAAACCTCCCTAGCACTCTGTGCTCGCTGGGGAAACAGGCAAGGCTGaagtctcccaggtatgcttgtttattgggtgtcatttttctttatagctgaacaaaattctactatatatacataccactggctctgttttctagtaactgagtataaactgactatattgctaatatgacgtttgcttcattttgtttagaaaatgggcagtgctttttgtaatgtcatctttctgaattatatctgaacaaggcttgttatgtttttcctacaccactagaatggataacttcagtttgagtgatgcgtattgttacagggactagtgacgttcaaagacgtggccattgattttacccaagaagaatggaagcaactggatcctactcagcggaacctatataggaatgtgatgctagaaaactacaacaacttaatcacagtaggtaagataatgtaccttttctaaaacagatgcaattgttaatgttgatgagccttgccttgaggtgccttgagatgttgctgtccctgccccagctttcgaaggaaaatctttggtggcattccagggtacaacattctcagaagttagtacacctggctctcccaggcccaactcagaatccagggttgtttttgtcactcgtgtgtatacccaaaccaatgtcatcccctcccccaccccccaggcccgccactcaccaaacctgaagtgattttcaagttggagcaagaggaagagccttgtgtggtggagagagaagtgctgtggagaccctgtccaggttagtgtgtgtgtgtgtgtggggggtgggggggacccagccaggtaaagcgagaggaccagtaagtgactggcctgagatgtttcctaaggtagattcccacagcgtgtagtaacagttatgtgaaactaagatgttctctttaaaaaaaaaaaaaaaattccctgcacttgggaggcagAGGCAGGTGGATTTCTGAGTTTGAGGCCAGCCTGGCCTACAAAGTGAGTTCCAGAACAGCCAGGGCTATGCAGAGAAACCCTGTCTCAGAAAAAAAATTTTTTTTAATTACGTTTTTTTGTGTACACACATGTG
Mutation details
JAX Protocol
Protocol Primers
| Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
|---|---|---|---|---|---|---|
| 49756 | GTG GCA TTC CAG GGT ACA AC | Wild type Forward | A | |||
| 49757 | CAC ACG AGT GAC AAA AAC AAC C | Wild type Reverse | A | |||
| 49758 | CCA AAA CCT CCC TAG CAC TCT | Mutant Forward | A | |||
| 49759 | AGG CTG GCC TCA AAC TCA G | Mutant Reverse | A | |||
| 49760 | Fluorophore-1 | CAC CTG GCT CTC CCA GGC C | Quencher-1 | WT Probe | ||
| 49761 | Fluorophore-2 | ACA GGC AAG GCT GAG GCA GG | Quencher-2 | MUT Probe |
Reaction A
| Component | Final Concentration |
|---|---|
| Kapa Probe Fast QPCR | 1.00 X |
| ddH2O | |
| 49756 | 0.40 uM |
| 49757 | 0.40 uM |
| 49758 | 0.40 uM |
| 49759 | 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.