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Notes
caaaatgcctttaaaatgacattctctttctttatagAGATACTTTCTAGTTGGGAGCAATCATGCAGAAACGAAGTACCGTGT(c/t)CTG
AAAA(t/c)TGATAGAACAGAACCGAAGGACTTGGTGGTAATCGACGACAGGgtgagtatcgttaggctccctgcccggccctcatgca
gggc
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 = T, C
Heterozygote = C/T and T/C
Wild type = C, T
Sequence
caaaatgcctttaaaatgacattctctttctttatagAGATACTTTCTAGTTGGGAGCAATCATGCAGAAACGAAGTACCGT
GT(c/t)CTGAAAA(t/c)TGATAGAACAGAACCGAAGGACTTGGTGGTAATCGACGACAGGgtgagtatcgttaggctccctg cccggccctcatgcagggcJAX Protocol
Protocol Primers
| Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
|---|---|---|---|---|---|---|
| 32990 | GCA ATC ATG CAG AAA CGA AG | Forward | A | |||
| 32991 | TGT CGT CGA TTA CCA CCA AG | Reverse | A | |||
| 32992 | Fluorophore-1 | CGT GTC CTG AAA ATT GAT AGA ACA G | Quencher-1 | WT Probe | ||
| 32993 | Fluorophore-2 | CGT GTT CTG AAA ACT GAT AGA ACA G | Quencher-2 | MUT Probe |
Reaction A
| Component | Final Concentration |
|---|---|
| Kapa Probe Fast QPCR | 1.00 X |
| ddH2O | |
| 32990 | 0.40 uM |
| 32991 | 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.