For in-depth product & services help, ask our
Technical Information Scientists
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= 118 bp
Wild Type = 130 bp
Sequence
Large deletion: Mutant sequence with junction in uppercase
tgttacatttacctaaattaccattctactgagctgaaacttaccaaaaccgtctactctgccatttttaaattccccctcaaaggtcatgttgtcatatcgaatgaagactccgacgccattgaacttgccctgggaaaactccccctcatacctgcagagacaccaagatgtaagttagcctttagtgcataggttagctgtgcggggtaacggaggccagcagctgtcaatgtgtctgttagtcagggtacagagggaagctcatctcagaactcttctagtgccacctggaccactcctcagtagcagagaagGGtggcagcttacagaagaagaggatgacagctctaagaaacagagagagaaagcagtgtcacggaatggctagtggactagaggcggtgctccggcaagtgcacacttacctcaagtggactcctacctcaggtcacttaaaacgccctcctcgccaccaccttcgcctctgctatggcaccctcacccctacttcttt
This allele was generated at The Jackson Laboratory by electroporating Cas9 protein and 2 guide sequences: ACCTTTGGTC AGGGTCATGG and GCTGCGCCCA AAGCTCCGTC. This resulted in a 1,823 bp deletion of Chr19:42,076,729-42,078,551 (GRCm38/mm10) that removes exon ENSMUSE00000373245 and/through ENSMUSE00001462078
JAX Protocol
Protocol Primers
| Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
|---|---|---|---|---|---|---|
| 79756 | TCA CCC TCT TTC CAC TCG | Wild type Forward | A | |||
| 79757 | CAC TGC TTT CTC TCT CTG TT | Common | A | |||
| 79758 | GGA AGC TCA TCT CAG AAC TC | Mutant Forward | A | |||
| 79759 | Fluorophore-1 | CGC TGG AGT AGG TAA AGG AA | Quencher-1 | WT Probe | ||
| 79760 | Fluorophore-2 | AGC TGC CAC CCT TCT CTG C | Quencher-2 | MUT Probe |
Reaction A
| Component | Final Concentration |
|---|---|
| Kapa Probe Fast QPCR | 1.00 X |
| ddH2O | |
| 79756 | 0.40 uM |
| 79757 | 0.40 uM |
| 79758 | 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.