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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
>chr9:44400104+44400175 72bp AGGACTAGCCTTTGCCTTGC CCACCCAATCTCCATTACCA
Mut= 106 bp
Wt= 72 bp
Fam=Mut
Hex=Wt
Sequence
Wt Sequence:
CTGCTGCTTATCCACAATGAACCTGCTGATGTTGGACTCCGAAATCTGGACCCTGCCCCCTCCAAGGGCAAAAAGGgtaagcctctatcaagctgactcctagagcaccttcctttgcccctggctccgcacgattttgttaggactagcctttgccttgccctgtgcttgccactggatgtaggcgaaggaatggtaatggagattgggtgggagaagtaaagattctatcctgaaatcagccagcgcatcctgagccttctctgacagcacagatctctccattctgggtaaccccctaagtttaagcagtttgggtgccttcttccacctgcaccgtcctcatttcctttcattt
Mutant Sequence:
CTCCGAAATCTGGACCCTGCCCCCTCCAAGGGCAAAAAGGGTAAGCCTCTATCAAGCTGACTCCTAGAGCACCTTCCTTTGCCCCTGGCTCCGCACGATTTTGTTAGGACTAGCCTTTGCCTTGCCCTGTGCTTGCCACTGGATGATAACTTCGTATAGCATACATTATACGAAGTTATTAGGCGAAGGAATGGTAATGGAGATTGGGTGGGAGAAGTAAAGATTCTATCCTGAAATCAGCCAGCGCATCCTGAGCCTTCTCTGACAGCACAGATCTCTCCATTCTGGGTAACCCCCTAAGTTTAAGCAGTTTGGGTGCCTTCTTCCACCTGCACCGTCCTCATTTCCTTTCATT
JAX Protocol
Protocol Primers
| Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
|---|---|---|---|---|---|---|
| 59439 | Fluorophore-1 | TG+ TA+ T+G +CT ATA +C+ GAA G | Quencher-1 | MUT Probe | ||
| 64036 | AGG ACT AGC CTT TGC CTT GC | Forward | A | |||
| 64037 | CCA CCC AAT CTC CAT TAC CA | Reverse | A | |||
| 64038 | Fluorophore-2 | CAC TGG ATG TAG GCG AAG | Quencher-2 | WT Probe |
Reaction A
| Component | Final Concentration |
|---|---|
| ddH2O | |
| Kapa 2G HS buffer | 1.30 X |
| MgCl2 | 2.60 mM |
| dNTPS-kapa | 0.26 mM |
| 64036 | 0.50 uM |
| 64037 | 0.50 uM |
| Glycerol | 6.50 % |
| Dye | 1.00 X |
| Kapa 2G HS taq polym | 0.03 U/ul |
| 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.