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Stock No: 008194
Protocol 32530: Probe Assay - Gata4<tm1.1Sad> 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= 143 bp
Wild Type = 143 bp
>chr14:63821675-63821817 143bp TCACATGGTGCTCTGAACTTG CAGATCCCAGTCAATAGCAATGT
Sequence
Wt Sequence: ttttcaagctttaatcaaagcagacaagttcccattagccgtgctcctgacagactgatctataatcggggtacatgtgtatcacatggtgctctgaacttgtacaagttccttcaGGGATCCtctcaagaccccacagaagcagtgtcatcctatcttcctgagaagcaaactgaaagtcaagtgcaatggcctaaaatcacattgctattgactgggatctggaatgttcactccgggatcaatagtctagtcaatataccgttctgttgtcacagtctctatgt
Mutant Sequence:AGGAAACCCTATGACGACTGCCCTTGCAATAGGCTGGTTCAATCAGTGTGGGGCCGGAGAGAGAAATTTTGCTCAGTGGTCCATTTTTCAAGCTTTAATCAAAGCAGACAAGTTCCCATTAGCCGTGCTCCTGACAGACTGATCTATAATCGGGGTACATGTGTATCACATGGTGCTCTGAACTTGTACAAGTTCCTTCAGGGATCtcgaggaattccgatcatattcaataacccttaatatAACTTCGTATAATGTATGCTATACGAAGTTATtaggtctgaagaggagtttacgtccagccaagctaGGATCCTCTCAAGACCCCACAGAAGCAGTGTCATCCTATCTTCCTGAGAAGCAAACTGAAAGTCAAGTGCAATGGCCTAAAATCACATTGCTATTGACTGGGATCTGGAATGT
JAX Protocol
Protocol Primers
| Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
|---|---|---|---|---|---|---|
| 39319 | TCA CAT GGT GCT CTG AAC TTG | Common | A | |||
| 39320 | GCT TGG CTG GAC GTA AAC TC | Mutant Reverse | A | |||
| 39321 | CAG ATC CCA GTC AAT AGC AAT GT | Wild type Reverse | A | |||
| 39322 | Fluorophore-1 | AGG GAT CTC GAG GAA TTC CGA TC | Quencher-1 | MUT Probe | ||
| 39323 | Fluorophore-2 | TGT CAT CCT ATC TTC CTG AGA AGC A | Quencher-2 | WT Probe |
Reaction A
| Component | Final Concentration |
|---|---|
| Kapa Probe Fast QPCR | 1.00 X |
| ddH2O | |
| 39319 | 0.40 uM |
| 39320 | 0.40 uM |
| 39321 | 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.