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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= 112 bp
Wild Type = 108 bp
Sequence
Large deletion: Mutant sequence with junction in uppercase
tctgggtgacagagctcaggcctaccctcctaactgccagcctgtctttacttattaggagagcagaggcccagggcctctgctctcccacttggtcccttgctggctagcaatgtgaagactagagtatggaaacagccttccgagtcttcacttgaaagtgaaggccatgcaggtctggggccacaaaatggctgctgctgctgtgacaaaacaattcctgaccaaccagcttctgcctaaggacttaggtttctgttgttgttcaaagcaggtgacagtagttaaagacatcatctgctgcaagagtaggagaagatGGccagctgggtcatagggaggcaggtgacagatgaaagctactccctgtctccactgagaactgctaagacaaggtcagagtttcccctgccattttcatggttggatctttaatgtcatctaactttttacacagcaattaaacaaccttgtgagagccagtgagagtggctacaattgatttcactggggctcttcttgtgttcaagcttccttttttatttttaaaaaagtactgtgcaccacatgtgcagaggccagaagagacctttggatccccttgggactggagttgcagacagttgttagctgccctgtgggtgctggcaactctacctgggtcctctggaagagaaggggcccctaacttccgagtcgcccctccagccctcttcattttctaa
This allele was generated at The Jackson Laboratory by electroporating Cas9 protein and 2 guide sequences: GCAGTTGGTA TGGTTCACAG and TATGACCCAG CTGGCCTGTG. This resulted in a 4,949 bp deletion of Chr11:103,134,082-103,139,030 (GRCm38/mm10) that removes exons ENSMUSE00000388880 and ENSMUSE00000377993
JAX Protocol
Protocol Primers
| Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
|---|---|---|---|---|---|---|
| 74191 | AGC AGA GCC ATC TTA GTT TG | Wild type Forward | A | |||
| 74192 | TGA GAT ATT TGC AGT GTC CC | Wild type Reverse | A | |||
| 74193 | TCT GTT GTT GTT CAA AGC AG | Mutant Forward | A | |||
| 74194 | GGA GTA GCT TTC ATC TGT CA | Mutant Reverse | A | |||
| 74195 | Fluorophore-1 | CCA GTA AGA CAA GGC CAA TT | Quencher-1 | WT Probe | ||
| 74196 | Fluorophore-2 | CTG GGT CAT AGG GAG GCA GG | Quencher-2 | MUT Probe |
Reaction A
| Component | Final Concentration |
|---|---|
| Kapa Probe Fast QPCR | 1.00 X |
| ddH2O | |
| 74191 | 0.40 uM |
| 74192 | 0.40 uM |
| 74193 | 0.40 uM |
| 74194 | 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.