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Stock No: 002106
Protocol 18226: End Point Analysis Assay - Tyr c-EP
Version 4.0
Notes
This mutation introduces a DdeI enzyme restriction site.
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 = C/C
WT = G/G
Het = C/G
Sequence
ATGTTCTTGGCTGTTTTGTATTGCCTTCTGTGGAGTTTCCAGATCTCTGATGGCCATTTTCCTCGAGCCTGTGCCTCCTCTAAGAACTTGTTGGCAAAAGAATG
CTGCCCACCATGGATGGGTGATGGGAGTCCCTGCGGCCAGCTTTCAGGCAGAGGTTCCTGCCAGGATATCCTTCTGTCCAGTGCACCATCTGGACCTCAGTTCCCCTTCAAAGGGGTGGA
TGACCGTGAGTCCTGGCCCTCTGTGTTTTATAATAGGACCTGCCAGTGCTCAGGCAACTTCATGGGTTTCAACTGCGGAAACT(g/c)TAAGTTTGGATTT
GGGGGCCCAAATTGTACAGAGAAGCGAGTCTTGATTAGAAGAAACATTTTTGATTTGAGTGTCTCCGAAAAGAATAAGTTCTTTTCTTACCTCACTTTAGCAAAACATACTATCAGCTCAGTCT
ATGTCATCCCCACAGGCACCTATGGCCAAATGAACAATGGGTCAACACCCATGTTTAATGATATCAACATCTACGACCTCTTTGTATGGATGCATTACTATGTGTC
Albino = C
JAX Protocol
Protocol Primers
| Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
|---|---|---|---|---|---|---|
| 17285 | AAT AGG ACC TGC CAG TGC TC | Forward | A | |||
| 17286 | GCC TGT GGG GAT GAC ATA GA | Reverse | A | |||
| 17430 | Fluorophore-1 | CTG CGG AAA CTC TAA GTT TGG AT | Quencher-1 | MUT Probe | A | |
| 17432 | Fluorophore-2 | CTG CGG AAA CTG TAA GTT TGG AT | Quencher-2 | WT Probe | A |
Reaction A
| Component | Final Concentration |
|---|---|
| Kapa Probe Fast QPCR | 1.00 X |
| ddH2O | |
| 17285 | 0.40 uM |
| 17286 | 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.