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Notes
Taqman Probe/Endpoint protocols use a different analysis than qPCR. Please see your equipment manual for more information about Taqman endpoint analysis setup.
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= 76 bp
Wild Type = 87 bp
Sequence
Large deletion: Mutant sequence with junction in uppercase:
ccccctccccaatcttaacacctacttgtgggctagtcccataaaggaatggccctcggctggaaggctttctggaacttagagtgcttggtcctttttaaaatcattttcgtttcctttgattcgtccttttcagggctttgcctgaaatggacaaccggaTCaaaaggaacggactgtgcactagagacttgcagcccacagccttccgtgtgggatctgccaggacaagctggctagacctctgagaagttggagaagtcaggaaattgtactctccac
This allele was generated at The Jackson Laboratory by electroporating Cas9 protein and 2 guide sequences: GGTAGTCATC ATAGGGAATC and CCCAGATTATCATCGCCAAA. This resulted in a 1,944 bp deletion of chrX:100,623,417-100,625,360(GRCm38/mm10) that removes exons ENSMUSE00001214740 through ENSMUSE00000696919
JAX Protocol
Protocol Primers
| Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
|---|---|---|---|---|---|---|
| 76018 | GAA GTT GAG AAC TCA TCC TGA | Wild type Forward | A | |||
| 76019 | CTA GTG CAC AGT CCG TTC | Common | A | |||
| 76020 | GTT TCC TTT GAT TCG TCC TT | Mutant Forward | A | |||
| 76021 | Fluorophore-1 | TGC TGT GTT CTT TCT TAC CC | Quencher-1 | WT Probe | ||
| 76022 | Fluorophore-2 | CTT TGC CTG AAA TGG ACA ACC G | Quencher-2 | MUT Probe |
Reaction A
| Component | Final Concentration |
|---|---|
| Kapa Probe Fast QPCR | 1.00 X |
| ddH2O | |
| 76018 | 0.40 uM |
| 76019 | 0.40 uM |
| 76020 | 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.