Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.
>chr8:92865160+92865264 105bp CATGAAATGTCTCCAATAGCTTC CTAGTCCTTTGATCAGGCACA
Mut= 96 bp
Wt= 105 bp
Fam=Mut
Hex=Wt
Wt Sequence (deletion in lower case):
GTGACACACCTACTCCAACCAGGTCACACCTATTCCAACAAGGCCACACCTCCAAATGGTGCCACTCCCTGGTCCAAGAATATACAAACCATGATAAATGTCCTATCTTGTGTACATATTTGTTTGTGTGTGTTTATATCTCAGTCTCATGCAAGGCATTTAATTAATTCACCcttgagaacatgcaggaaaccaaaaactttcctgttacttacttttttttttttttttttttttttttttttggtcaactttggtttgcagatgtttcttcttggaattattctgctcccagtccgtgctttactggttggaataattttgctgctcgcatggccatttgctgtcatttcaacagcatgctgtcctgaaaagctgacccatccaataagcaattggaggaggtaagtggtaatcatgtccaaatattacacaaatcaaaacttttaaagtttctccaatgaccagtttttgtgaaggatattcatctttggttctaaagagcccctgggatatgttttaacatgaaatgtctccaatagcttcatgcattagaggctttattgacagccaatgggCTTTTGGGGAGTGACTGTATCTTGAGGGCTGTGCCTGATCAAAGGACTAGTGCCTTTATGGATTCATAATATGATGGCATTATAAGGAGGTGGTGAAAATCGGGGGGGGGTAGCCTAGTTGGAATAATTATGTCAATTACTAGGAGTATGTCCTG
This mutation is a 405 bp deletion beginning at Chromosome 8 position 92,864,810 bp and ending after 92,865,214 bp (GRCm38/mm10).
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
45596 | CAT GAA ATG TCT CCA ATA GCT TC | Wild type Forward | A | |||
45597 | CTA GTC CTT TGA TCA GGC ACA | Common | A | |||
45598 | GTG TGT TTA TAT CTC AGT CTC ATG | Mutant Forward | A | |||
45599 | Fluorophore-1 | CAT TAG AGG CTT TAT TGA CAG CC | Quencher-1 | WT Probe | ||
46568 | Fluorophore-2 | AAT TAA TTC ACC CTT TTG GGG A | Quencher-2 | MUT Probe |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
45596 | 0.40 uM |
45597 | 0.40 uM |
45598 | 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 |