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Stock No: 031948
Protocol 36331: End Point Analysis Assay - Slc6a17<em1Adiuj>
Version 1.0
Notes
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 = G/G
Heterozygote = A/G
Wild type = A/A
>chr3:107500432-107500497 66bp GAGGAGCTGGACACAGAGGA AATCTGGGCCAGGATGTACT
Sequence
GTCAGTGGCTGACTTGCTGGCTCTGGAGGAGCCTGTGGACTACAAGCAGAGTGTACTGAACGTGGCCGGCGAGACAGGTGGCAAGCAGAAGGTGGCGGAGGAGGAGCTGGACACAGAGGACCGGCC(a/g)GCCTGGAATAGCAAGCTGCAGTACATCCTGGCCCAGATTGGCTTCTCTGTGGGCCTGGGCAACATCTGGAGGTTCCCCTACCTGTGCCAGAAAAATGG
JAX Protocol
Protocol Primers
| Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
|---|---|---|---|---|---|---|
| 48458 | GAG GAG CTG GAC ACA GAG GA | Forward | A | |||
| 48459 | AAT CTG GGC CAG GAT GTA CT | Reverse | A | |||
| 48460 | Fluorophore-1 | CGG CCA GCC TGG AAT AG | Quencher-1 | WT Probe | ||
| 48461 | Fluorophore-2 | CGG CCG GCC TGG A | Quencher-2 | MUT Probe |
Reaction A
| Component | Final Concentration |
|---|---|
| Kapa Probe Fast QPCR | 1.00 X |
| ddH2O | |
| 48458 | 0.40 uM |
| 48459 | 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.