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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
>chrX:56913179+56913269 91bp CAAATGTTTCACAGCAAATAGACC GGCAACAGTGTGGATGAATG
Mutant= 111 bp
Wild Type = 91 bp
X-Linked
Sequence
Wt Sequence (deletions in lower case; insertions with carrots ^g^ (g insertion):
CAAATGTTTCACAGCAAATAGACCTAACAACTTCTTCCCAA^g^actactggacttaacccacacaagacttcacattcatccacactgttgccagaaggcatggctgattctacaatcaacagtacagccatatcatatgctaatacagtaccctcacctctagcaacagtaagtgcagccaaagatttgaagacatctacaaccgagacagctacatttttaacagataccttgtttacttcaacagccacccctctgcctacccagactgtaacagaacactcatatctaggaaaaacaaggacatcaaaaagggtggaggccatggctactgagatctttcattcagctacagctactgatttaattgacacatctgtgtttactaagaattatactgtatctgaaacttcaacaactaagtccaagtcagctgttgggaagacaactttattcttgaatgaatcaacatccatagctccaacaccttgtcccaagcacaaatccacagatgttgctatactccatacctctaaatctgggcaagaatttcttgtatcttcagctgccagaactgtgtcttggtccacattagaagagacttcacctattactactgatgttgggattgtatcaaccttcccaccagagtctttgcttacctccacagcttctcctgtgagttctacatttcctgaaattcagttagcatctaccttgtcaacaactgattcagaaatggcatcaacagtccattcagtgttgcctatgcagaccatacctaccccaagaacagtgaaaccagagtcaggctctacaaatttccaagatgtcttttcacccagcatggaggatgctctatctgtctccatgcctaaagaaaccactttcatggccttttcttccattacctcctctccaatcaccagaactcaagatgaacagatagctattgatgctgagagtacacatctaaccataatccccggaaccaaatttgtccctacattggctgaagcttcactcttccctaccattgaagggcaagcatacactcaggatacacccacaacggatgagcccatgcttactttgacttctactaaatcaccttctacatataacgcatctgaatcagttctcacatctataacaattaaaagtgactatcagttcttcaccaatgagaccacttggacttctaagtcaggtcagaacctgttgacctctatgaatacaaccactatacccacattcacatctaataaaactctcacattaccattccaaggtaatgccactaatagagatcattcatccatgactactaacgtctcaccaattgaagcatccacggagagcaaagtgactaccagctctgatgctactacagccagttatacaacagctttattcaaaccaacatcccagtggcttagtcatttcacgagtgtttctggaatcacatctatagccagccagcctgaatctaaactcaccaccttattattgaaaagcaactctatgcccacagtagctacaaatgaatttccctcaataccaagtgagccagttgctccatcagtgaatacgtctaccctcactgacattaaaccaaatttttctacagagaagagcatttctgagacaatacaaatagaaacaaatggtgtgagctcatttggtgatactctggcccctctgcccatgtctgcaacaacacagagagtctataccactgtgacaaaagaaaccacttcccgtcatcctaaggtaaagtcaactatatcaacagtagctgaggcttctccattttcaacaatgctggaagtgacagatgaatcagaacaaatggtgacagcttctgtcactatttccccctttacagatatagaaaaattgactactgcattgagtaaagaaactgcaactgcagaggtgggagtgagttggctttcaaccaaattaaaggaaagcatgcctgagagttcacacaatggaaccacagaaagctttaactcgacccatacctacacagtagattggacttcagaaaaatccaagggtaactcagcttcatcacccaattctgcaagcacccaggcactacctgaacttccaagttcttcaacaatgaagacgatgggagtcactttctccactaattcttcacagaggacagctgcatctttgtctgctggtatcttgtctccacagactgcctctactcatgccttagtaactccccaactgcttacaca
This mutation deletes 5813 bp of ENSMUSE00000386381 (exon 3) and is predicted to cause a change of amino acid sequence after residue 254 and early truncation 9 amino acids later. There is a 1 bp insertion (G) at the deletion site.
JAX Protocol
Protocol Primers
| Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
|---|---|---|---|---|---|---|
| 49531 | GAG AAC ATG GAG AAC ACA TGG A | Mutant Reverse | A | |||
| 49532 | CAA ATG TTT CAC AGC AAA TAG ACC | Common | A | |||
| 49533 | GGC AAC AGT GTG GAT GAA TG | Wild type Reverse | A | |||
| 49534 | Fluorophore-1 | CTA CTG GAC TTA ACC CAC ACA AGA C | Quencher-1 | WT Probe | ||
| 49535 | Fluorophore-2 | CAA CTT CTT CCC AAG TAC AGG TAT TT | Quencher-2 | MUT Probe |
Reaction A
| Component | Final Concentration |
|---|---|
| Kapa Probe Fast QPCR | 1.00 X |
| ddH2O | |
| 49531 | 0.40 uM |
| 49532 | 0.40 uM |
| 49533 | 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.