Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.
Mutant= 96 bp
Wild Type = 78 bp
Wt (NOD background) Sequence: GACCGCGGGGTCCCGCAGGCTCTCACACGTTCCAGCGGATGTTCGGCTGTGACGTGGGGTCGGACTGGCGCCTCCTCCGCGGGTACCAGCAGTTCGCCTACGACGGCCGCGATTACATCGCCCTGAACGAAGACCTGAAAACGTGGACGGCGGCGGACACGGcggcgctgatcaccagacGCAAGTGGGAGcaggctggtgatgcagagtattacAGGGCCTACCTAGAGGGCGAGTGCGTGGAGTGGCTCCGCAGATACCTGGAGCTCGGGAATGAGACGCTGCTGCGCACAGGTGCAGGGGCCGCGGGCAGCTCCTCCCTCTGCCCTCGGGCTGGGGCTCAGTCCTGGGGAAGAAGAAACCCTCAGCTGGGTGATGCCCCTGTCTCAGAGGGGAGAGAGTGTCCCTGGGTCTCCTGATTCCTCATCACAGGGACTGCACTGAGTCTCCCAGGACTCAGCCCTCTCCCTGGACAGTGCCCAGGCTGTCTCAGGAGGGAAGGAGAGAATTTCCCTGAGGTAACAACAGCTGCTCCCTTCAGTTCCCCTGCAGCCTCTGTCAGCCATGGCCTCTCCCAGGCCGG
Mutant Sequence: ctttgctttccCtggttaagcagtacagccccaaaatggttaaggttgcaagCTTGCTGGTGAAAAGGACCTCTCGAAGTGTTGGATACAGGCCAGACTttgttggatttgaaattccagacaa
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
49452 | AGC AGT ACA GCC CCA AAA TG | Mutant Forward | A | |||
49453 | CTG GCC TGT ATC CAA CAC TTC | Mutant Reverse | A | |||
49454 | Fluorophore-1 | AGG TTG CAA GCT TGC TGG TG | Quencher-1 | MUT Probe | ||
50991 | CTG AAC GAA GAC CTG AAA ACG | Wild type Forward | A | |||
51486 | CCT GTA ATA CTC TGC ATC ACC AG | Wild type Forward | A | |||
51487 | Fluorophore-2 | CGG CGC TGA TCA CCA GAC | Quencher-2 | WT Probe |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
49452 | 0.40 uM |
49453 | 0.40 uM |
50991 | 0.40 uM |
51486 | 0.40 uM |
Wt Probe | 0.15 uM |
Mutant Probe | 0.15 uM |
DNA |
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 |
Stock Number | Strain Name |
---|---|
025216 | NOD.Cg-Prkdcscid H2-K1b-tm1Bpe H2-Ab1g7-em1Mvw H2-D1b-tm1Bpe Il2rgtm1Wjl/SzJ |
023848 | NOD.Cg-Prkdcscid H2-K1b-tm1Bpe H2-D1b-tm1Bpe Il2rgtm1Wjl/SzJ |
037275 | NOD.Cg-Rag2em8Lutzy H2-K1b-tm1Bpe H2-Ab1g7-em1Mvw H2-D1b-tm1Bpe Il2rgtm1Wjl/J |
3 strains use this protocol |