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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= 74 bp
Wild Type = 103 bp
Sequence
Large deletion: Mutant sequence with junction in uppercase
actgaggcccaaccccgcagtccaggtagggggaaggggatcagtatgttgattaggagggttttgaacttgtagtgacatccctcctgctgagattataggcatactccatgcacgcccagctaaatagattagctgtttgttggacgccttctcctctcttgggagtcctttctcacttttccttaataatcctaaactcactctgctaaaaaaaaagaaaaaaagaaaaagtaaaagaaagagtgtaaggaaaaactttggcaagTAgtgctgttgggcatcccaggggattgatggtgttagctatgtttatttcctatctatcaacttagttcctgtctccacatggaagatcactgaaaactgccttatattagttaactgggtgagtgatgggatcttgaggtcccaaagttccagtgtgagctaaataggtcagctggagggagagctgtgaatatcctggcagtccagagcccgggataaggggatggagagacacacttctaataagcccaaaagaaaaaaagaaaatgtaagcagagggactcctagataattctcaaaacgaatttaggatcttttcactctttgccacatcttctctgggtttttaagcctattctggatcctggacttagtgatgtgtagtttctg
This allele was generated at The Jackson Laboratory by electroporating Cas9 protein and 2 guide sequences: GGATGCCCAACAGCACTCAC and GAAAAACTTTGGCAAGTTGC. This resulted in a 1,453 bp deletion of Chr11:48,825,274-48,826,726 (GRCm38/mm10) that removes exon ENSMUSE00000665110.
JAX Protocol
Protocol Primers
| Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
|---|---|---|---|---|---|---|
| 76857 | AAG AGA TCA GAC CGA CGT AT | Wild type Forward | A | |||
| 76858 | GTT TCA GCC AAG CAC TCC | Wild type Reverse | A | |||
| 76859 | AGT AAA AGA AAG AGT GTA AGG A | Mutant Forward | A | |||
| 76860 | CTA ACA CCA TCA ATC CCC TG | Mutant Reverse | A | |||
| 76861 | Fluorophore-1 | TGA ATG ATG CCT CGT TTT AGC | Quencher-1 | WT Probe | ||
| 76862 | Fluorophore-2 | AGT AGT GCT GTT GGG CAT CC | Quencher-2 | MUT Probe |
Reaction A
| Component | Final Concentration |
|---|---|
| Kapa Probe Fast QPCR | 1.00 X |
| ddH2O | |
| 76857 | 0.40 uM |
| 76858 | 0.40 uM |
| 76859 | 0.40 uM |
| 76860 | 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.