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Stock No: 004525
Protocol 21385: Probe Assay - Bcl2l11<tm1.1Ast> -Probe Alternate2
Version 1.0
Notes
Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination. The transgene genotype is determined by comparing ΔCt values of each unknown sample against known homozygous and hemizygous controls, using appropriate endogenous references.
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= 100 bp
Wild Type = 96 bp
Sequence
Wt Sequence:gtcaagggaccctgagatgtttctgtctttacatagacatctcccgtggagctaccggcccacactaagtggctgctccccagactgcttgagcagccttgttccttttgagtagcaGATCTatcagggagtcccggatctcgagaataatctcagccccacttcctgctcttctgtgggacgtgttgggggacgaatgtgtgtgctatagtctggcttctgtcataatgggcac
Mutant Sequence: tcgacggtatcgataagcttgatatcgaattcctgcagcccggggGATCTATCAGGGAGTCCCGGATCTCGAGAATAATCTCAGCCCCACTTCCTGCTCTTCTGTGGGACG
JAX Protocol
Protocol Primers
| Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
|---|---|---|---|---|---|---|
| 32330 | GAT AAG CTT GAT ATC GAA TTC CTG | Mutant Forward | A | |||
| 32331 | CGT CCC ACA GAA GAG CAG | Common | A | |||
| 32332 | CTT GAG CAG CCT TGT TCC T | Wild type Forward | A | |||
| 32333 | Fluorophore-1 | CTT TTG AGT AGC AGA TCT ATC AGG GAG | Quencher-1 | WT Probe | ||
| 32334 | Fluorophore-2 | AGC CCG GGG GAT CTA TCA | Quencher-2 | MUT Probe |
Reaction A
| Component | Final Concentration |
|---|---|
| Kapa Probe Fast QPCR | 1.00 X |
| ddH2O | |
| 32330 | 0.40 uM |
| 32331 | 0.40 uM |
| 32332 | 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.