Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.
>chr7:3675526-3675632 107bp GTTCCCAGTTTGTAGGTTCCA CCTGGATTCAGAGCCAGAAG
Mut= 97 bp
Wt= 107 bp
Fam=Mut
Hex=Wt
Wt Sequence (deletion in lower case):
CAAGTGCTGGGATTAAAGGTGTGCCCCCCCCCCCACCACCCAGCTCAGATTTGGTTCCTTTTTAATTGCTAAAGAGTATTCCAGTTTATGGATACAGTACCACGATGCCTGTCCTCGTTACATCGTGTTGTTTCTGTCTTTGGGTGGTTCCCAGTTTGTAGGTTCCAAACCCTTCAttttctgacgctgactctgctctctttctaggaagagccaggctacagggcagctggcttctggctctgaatccagggctgcctactggacccgacagctttcccggaccaagggaaaaatgaaagaaggctttcaaactatacagccctgggcatggacgctgaagaaaattgggggtgtgctgggctttagaggaagaggcctggggtctgaggctggagtgtggtttcctgggtctgacagagaggggagtggcaattatcccctgggcctttgGGAAAAGGTCTGGTTCCTGCAGCCCTGGTCTGAAGGATGAAGACTAGTGCCTGAACCCTTCTGAATCAGAGAGAAGAGAACTGGAATCTGAGGGGGGAGGGCTGGTTTCTGGACCCCAGGGTCAGACCAGGAGTCAGGGCTCTGTTATAGATTTCTGGGTCTGAGGGAGGAGGGCTATGGCCAGGACTCTGGGTTGGAACTACACTGGTAAGGGACCTGAGGCCT
This mutation is a 277 bp deletion beginning at Chromosome 7 position 3,675,326 bp and ending after 3,675,602 bp (GRCm38/mm10).
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
40951 | GTT CCC AGT TTG TAG GTT CCA | Common | A | |||
40952 | CCT GGA TTC AGA GCC AGA AG | Wild type Reverse | A | |||
40953 | GAT TCA GAA GGG TTC AGG CAC T | Mutant Reverse | A | |||
40954 | Fluorophore-1 | AGC CAG GCT ACA GGG CA | Quencher-1 | WT Probe | ||
40955 | Fluorophore-2 | TCA GGA AAA GGT CTG GTT CCT | Quencher-2 | MUT Probe |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
40951 | 0.40 uM |
40952 | 0.40 uM |
40953 | 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 |