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Stock No: 025692
Protocol 36546: Probe Assay - Vim<tm1Cba> 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= 109 bp
Wild Type = 109 bp
>chr2:13574579+13574687 109bp CGCAGCCTCTATTCCTCATCC AGTCCACCGAGTCTTGAAGC
Sequence
Wt Sequence: ATGTCTACCAGGTCTGTGTCCTCGTCCTCCTACCGCAGGATGTTCGGTGGCTCCGGCACATCGAGCCGGCCCAGCTCCAACCGGAGCTATGTGACCACGTCCACACGCACCTACAGTCTGGGCAGCGCACTGCGCCCCAGCACTAGCCGCAGCCTCTATTCCTCATCCCCCGGtggcGCCTATGTGACCCGGTCCTCGGCAGTGCGCCTGCGGAGCAGCGTGCCGGGCGTGCGGCTGCTTCAAGACTCGGTGGACTTCTCGCTGGCCGACGCCATCAACACTGAGTTCAAGAACACCCGCACCAACGAGAAGGTAGAACTGCAGGAGCTGAATGACCGCTTTGCCAACTACATCGACAAGGTGCGCTTCCTCGAGCAGCAGAACAAAA
Mutant Sequence:ACACGCACCTACAGTCTGGGCAGCGCACTGCGCCCCAGCACTAGCCGCAGCCTCTATTCCTCATCCCCCGGTGGCGtggttaccatggagccaaaaaagaagagaaaggtagaagaccccgtaccggtgggtgaagaccagaaacagcacctcgaactgagccgcgatattgcccagcgtttcaacgcgctgtatggcgagatcgatcCCGTCGTTTTACAACGTCGTGACTGGGAAAACCCTGGCGTTACCCAACTTAATCGCCTTGCAGCACATCCCCCTTTCGCCAGCTGGCGTAATAGCGAAGAGGCC
JAX Protocol
Protocol Primers
| Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
|---|---|---|---|---|---|---|
| 21265 | AGT CCA CCG AGT CTT GAA GC | Wild type Reverse | A | |||
| 49056 | CGC AGC CTC TAT TCC TCA TCC | Common | A | |||
| 49057 | CGA GGT GCT GTT TCT GGT CT | Mutant Reverse | A | |||
| 49058 | Fluorophore-1 | CCG GTG GCG TGG TTA CCA T | Quencher-1 | MUT Probe | ||
| 49059 | Fluorophore-2 | CCG GTG GCG CCT ATG TGA | Quencher-2 | WT Probe |
Reaction A
| Component | Final Concentration |
|---|---|
| Kapa Probe Fast QPCR | 1.00 X |
| ddH2O | |
| 21265 | 0.40 uM |
| 49056 | 0.40 uM |
| 49057 | 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.