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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
>chr4:140610610-140610704 95bp TAGAGAGGGGCCAAGGAAAG CCCAGGAACTGAGCCTATTC
Mutant= 100 bp
Wild Type = 95 bp
Sequence
Wt Sequence (deletions in lower case; bp changes in brackets with wt first and insertions with carrots ^g^ (g insertion):
TCTGACTGAGGTTGGGCCATGGGGGCGGTGTGGGGCAGGCAGGACAGGTAGGGAACTTACTCACTAGAGAGGGGCCAAGGAAAGGCAAGGGATTCTATAGGCACCCTGgttccttcgaggggcggggaacagggggtgggaataggctcagttcctggggccaacctctgtctcacaagctctctctctttgctcttcccacagacctggacccagcagctgcaccaccccagacagagtaagtgagctttgtccactgggaaaagagtagagcccaacaggggcgtgggacagggtaggacagtgactagagaatactgttgtttgttaagaccctttgcctggacgaggttggccctgaggctcgctgtggtggcctctttttctcttttgcctgggagcattgcattgcattgcctggtcccagacccacattcaggcctctccctacccatgtggcctcagtggactcacagactctcccagatttcctgtgttctgacttgaaacggaagggagagGAGACTCGCTTCCCTGGAGTTATCCCTGGGAACTAAGATGCTGTGGGATTGCTTCTGCTGATACCAGTCCTTCTCTTTCAAATTTTTAAATTACAAAATTTTATATTTATTGTGTGTGTGAGCATGCACGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTTTGAGTGTTCGAGTG
This allele was generated at The Jackson Laboratory by electroporating Cas9 protein and 2 guide sequences TGTTCTGACTTGAAACGGAA and CCCGCCCCTCGAAGGAACCA, which resulted in a 413 bp deletion beginning at Chromosome 4 position 140,610,248 bp and ending after 140,610,660 bp (GRCm38/mm10). This mutation deletes ENSMUSE00000335941 (exon 4) and 379 bp of flanking intronic sequence including the splice acceptor and donor and is predicted to cause a change of amino acid sequence after residue 74 and early truncation 43 amino acids later.
JAX Protocol
Protocol Primers
| Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
|---|---|---|---|---|---|---|
| 46646 | TAG AGA GGG GCC AAG GAA AG | Common | A | |||
| 46647 | CCC AGG AAC TGA GCC TAT TC | Wild type Reverse | A | |||
| 46648 | AGA AGC AAT CCC ACA GCA TC | Mutant Reverse | A | |||
| 46649 | Fluorophore-1 | TTC CTT CGA GGG GCG | Quencher-1 | WT Probe | ||
| 46651 | Fluorophore-2 | CAC CCT GGA GAC TCG CTT CC | Quencher-2 | MUT Probe |
Reaction A
| Component | Final Concentration |
|---|---|
| Kapa Probe Fast QPCR | 1.00 X |
| ddH2O | |
| 46646 | 0.40 uM |
| 46647 | 0.40 uM |
| 46648 | 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.