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Stock No: 008533
Protocol 34994: Probe Assay - Tg(Cspg4-cre)1Akik-Chr1-Probe
Version 1.0
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
Mutant= 82 bp
Wild Type = 102 bp
This assay may be capable of distinguishing hemi from hom. Transgene insertion site is known to be on mouse Chr 1.
This assay is designed around this insertion site, but it has not been tested on hom animals.
This assay is NOT able to be used for copy number evaluation. If this is required, it is suggested to type by qPCR.
Sequence
Wt Sequence:
gtagtccaaggatagcttatgctgcagcaatgaagggacagaaattcagcgcctatgtcagaaatacgggaatgaagctacatgtttcagttgtggAAaactgggacacccgagaaaggattgtaaaaatccctcaggaaacaagaagggccttcctccaggtccctgaccatgctgtggtaaaggaaagcactggaga
Mutant Sequence:
AGACCAATCCATAAACCAGGACCCTTACAGGATTATGTTAAAGCATGTATAGATGCTTCATGAACTGTAGTCCAAGGATAGCTTATGCTGCAGCAATGAAGGGACAGAAATTCAGCGCCTATGTCAGAAATACGGGAATGAAGCTACATGTTTCAGTTGTGGAAAACCACTTCCTGTGTATTCCTCTGTATTTGGGTACAGTCTCCAAGAAGCCTTCAAAACCTGCCTGGGCCAGGTTCATCCTGTTGTAGCCAACACCCATTCCTATACACTGAGAGAACACACAGACCATTTATAGATAAGCCACTTGAGCATGGGCAAGTCAGCAAGTCACAGGGGGATATCTGGGGGCAGGTTCTAGTACCAAATAAAGACAGTGTCAAC
JAX Protocol
Protocol Primers
| Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
|---|---|---|---|---|---|---|
| 44985 | CGG GAA TGA AGC TAC ATG TTT | Common | A | mChr1 | ||
| 44986 | CAG GGA CCT GGA GGA AGG | Wild type Reverse | A | mChr1 | ||
| 44987 | Fluorophore-1 | AGT TGT GGA AAA CTG GGA CAC | Quencher-1 | WT Probe | mChr1 | |
| 44988 | GGC TTC TTG GAG ACT GTA CCC | Mutant Reverse | A | mChr9 | ||
| 44989 | Fluorophore-2 | TGG AAA ACC ACT TCC TGT GTA TTC | Quencher-2 | MUT Probe |
Reaction A
| Component | Final Concentration |
|---|---|
| Kapa Probe Fast QPCR | 1.00 X |
| ddH2O | |
| 44985 | 0.40 uM |
| 44986 | 0.40 uM |
| 44988 | 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.