Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.
>chr12:111425084+111425173 90bp GAGAGGAGGTAGCTGGTGGA GGAAGTCAGGACTGAAAAAGCA
Mut= 90 bp
Wt= 90 bp
Fam=Mut
Hex=Wt
Wt Sequence (deletion in lower case):
GATTTCATTACGGGCGGTTGTGAGCCACCATGTGGTTGCTGGGATTTGAACTCAGGACCTCTGGAAGACCAGCCAGTGCTCTTAACTGCTGAGCCATCTCTCCAGCCCCATGACTTTAGACTTTAAGCCAGGTTAGAGAGAGGAGGTAGCTGGTGGAGCATCAGAGGTCCGCCTGGgtcctaacaactccctcatcccggaatcttgctttttcagtcctgacttcctgggcgccccagacttggcgctgcccaccgagccgctgcccccacttctagagcctgctctgtgggctcgactggaaagtgactacactagcttcctagaggtgaggctggggaggccccgggctccagaggttgggtcctacaggaggtttgatgtggaggataggggttgtgctggcagtggtcctgtaagcgatggaccatgactggagtccggtgacgctagtccttgaaggcgcaaaatggagtttggtaggcagggggcgcccttaccttcctcctggaaagcttctcaccacaggccactcctaaccctacagaccaagatcacaagctgttttgacagcatcttgcagctggaacagaatcgctgggaagccgacgaggaccgggaagtactgcagggcctctaccacgcgcccctgtccatcgatgtgcacatggtgcgcccgggagggggtgaaggaagggctatgttcaggactcaggctcatgtcggagatccctcagagctaagttttactgagagaggaacctgtggAGTGATCTAGACTCTTGGGGCACATGGCCAGGTTAAATCACAGCTCTCGGGTCTGCCGGTGAATTCTAGTAGCTGTGTGGAAGGGGGGGAGGTGACTCTCACATTGGGGCCACTGAAAACCTGCAGCGATGGGGTGGGCTGGGTTCGGGACTCAACTGCTCTCAACATGTGGCATCCCTGGCAGCTGGTAGCTGAG
This mutation is a 592 bp deletion beginning at Chromosome 12 position 111,425,123 bp and ending after 111,425,714 bp (GRCm38/mm10).
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
46828 | GAG AGG AGG TAG CTG GTG GA | Common | A | |||
46829 | GGA AGT CAG GAC TGA AAA AGC A | Wild type Reverse | A | |||
46830 | CCC GAG AGC TGT GAT TTA ACC | Mutant Reverse | A | |||
46831 | Fluorophore-1 | AAC TCC CTC ATC CCG GAA | Quencher-1 | WT Probe | ||
46832 | Fluorophore-2 | CCG CCT GGA GTG ATC TAG ACT | Quencher-2 | MUT Probe |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
46828 | 0.40 uM |
46829 | 0.40 uM |
46830 | 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 |