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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= 109 bp
Wild Type = 132 bp
Sequence
Wt Sequence (deletion in UPPER case):
tgaagtcaccaaaattacacacctttcagattgttcaaagatctgactcatgggagagccaggccatcatctgaaggcaggacaagtttgtgctacaggtcatggctgatgcccagtggagtgccagttcatctcttgtcaggacagttttgaccccaagaagctgggaggctgcagtgaccctgtacacacttgttctttccattcagtggatgctaccatccatctttcacgggtatcaggctagtgtgttcctttagggacttccactaaaggtaggaaactgagatgaaaatgaggaaaccccGTGGAAAGTTCTACAGGTATCATGGTTCACTGCCTGTGCCGATCCACCTGACTTACTGTCAGTGAGCGTGATTTATGTCCTTTCCAGGGTGGGACCATCATCGGTAGTGCGCGCTGCAAAGCCTTTCGAAGCCGTGAAGGGCGCCTGAAAGCTGCCTGTAACTTGGCGCGCTTGGGCATAACCAATCTGTGCGTGATCGGCGGGGACGGAAGTCTCACGGGAGCCAACCTCTTCCGAAAGGAGTGGAGCGGACTTCTGGAAGAGCTGGCTCGGAATGGTAGGTTTCTCTTTCTGTTTCCACCCTTGTTAGTGCCTTGCTCTGAGTAGTCGGCCTCAGGTTTCTCTGTTGGTGCTTTTTAAGAGCTTCGGAAGTAACACATTGTGCCTTCTTTAAGGGGGAGGGGGGGACGACAGTTATTTTATTTATAGCTGACATGGACATACATACTTTTGAGCCATTTTTTAAAATTTCGTTTTAGTTTCTGAGATGTGGTCTCACTGTACTGACCAGCCTGGCATGTGAACTCATAGATCTACCTGTACCTGCCTCCTGCCAGTTTGGCCTTCAAAGTGTGTATCACCAGTTCCAGCTTCATTTTTAGTCATAGCCAAATTGTGGCTGTTGTTTTGCCTCCTATCATCTGGTACCACACAGTTTAAATAAGAACAGAAATGACAGTAAGCCCTACTGTGTGCTAGGAAATGCCCCCTTTCCTAGAGAGGCCTGTAAAACCCTCATGACAGTAATCATGTACACCCCCATCTAAGAGAAGAGTCCCAGGTCGTAGGGGAAGCCAGGCTGCTGTAACCGTCATCTTTGGGCATACTGGTCCTTGGATGCCAGCATCCATATTTCTAGCCATCATGTTGTATTTCTGCTGATAAGCTAGGATGTCTCTAAATCTATATGTTATAAGAGTTTAATTAAAAGTTGCTGATCTGCATTCTCTCTCTCTTAATTTAAATTTCTTTTGGTATTATTTAGGATGTCTGAGGATTCTATGTGAACTCTTGGCCCTGTCTCTTTAAAGCTCTTTTGATTATAATTCCAATTCTGTATCAAATGTCATCAGCAAGAGACCTTGCCATGTCTCCTCCTGGATCCGAGGTGTGCTTCTTGTCTCCCATACAGGTGATATCGATAACGACACAGTGCAGAAGTATTCCTACCTCAACGTGGTGGGTATGGTGGGCTCCATTGACAATGACTTCTGTGGCACAGACATGACCATTGGTACAGATTCAGCCCTGCACCGAATTATTGAAGTTGTTGATGCCATCATGACCACTGCTCAGAGGTAAAAGGACTTAAGGACACCAACAGGCTATTACTGCTGGTTCCAGAAACACTCAGCAGATCCCATGTTAGTAGTATTCTAAATGTGCTTATGGGAGAAAAACTAGAAAACACAAGGAAACAAAATAATTATCTAGAGTCCCCCAAAGgttatctcatggaatgacaaagagagagtgacttagtagcatagttgttagtggagtgagacatcctagtgtcaccattgagattccaaggtctcagtgagtagactgtgagtagaacgttaacagtttatctgcagtgatttaggcccacaaggatgcatggccgacaactttctagagtaccacagaattcccaaaagcctttccctgaaaatcaagtgaaatagtaagcctgacaggcacctttcactgtgtcagtggttagcttgaagcttgaagcattggaacaggtgtttttgagggcagctgggccttcgtgggtcccttttgtgtatcatgcctagtttttagctgcctgtgtccattgagctctgtgactctggaaggtcagctcagagcatttgactctttcaactgcaaggaatgacagtca
This allele was generated at The Jackson Laboratory by electroporating Cas9 protein and 2 guide sequences: AATTATCTAGAGTCCCCCAA and TACCTGTAGAACTTTCCACG. This resulted in a 1,446 bp deletion of Chr13:6,620,738-6,622,183 (GRCm38/mm10) that removes exons ENSMUSE00001255658 and ENSMUSE00001265994.
JAX Protocol
Protocol Primers
| Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
|---|---|---|---|---|---|---|
| 74212 | GAC TTC CAC TAA AGG TAG GA | Mutant Forward | A | |||
| 74213 | TGT CTC ACT CCA CTA ACA AC | Mutant Reverse | A | |||
| 74214 | Fluorophore-1 | CTG CTG TAA CCG TCA TCT TT | Quencher-1 | WT Probe | ||
| 74215 | Fluorophore-2 | TGG AAT GAC AAA GAG AGA GTG ACT | Quencher-2 | MUT Probe | ||
| 75385 | AAC CCT CAT GAC AGT AAT CA | Wild type Forward | A | |||
| 75386 | TGG CTA GAA ATA TGG ATG CT | Wild type Reverse | A |
Reaction A
| Component | Final Concentration |
|---|---|
| Kapa Probe Fast QPCR | 1.00 X |
| ddH2O | |
| 74212 | 0.40 uM |
| 74213 | 0.40 uM |
| 75385 | 0.40 uM |
| 75386 | 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.