Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.
>chr13:56450547+56450694 148bp CCCCAAGACCATGAACTCAG TCCCAACACATCTCCTCACT
Mut= 146 bp
Wt= 148 bp
Fam=Mut
Hex=Wt
Wt Sequence (deletion in lower case):
TCCCTTTGTCAGCCTGTCACCACAGACTTAGTGAGTTGCACAGTGTTGCCTGTCAGTTTGTCTAAACCCTCCATCCTGTCCTCATCTGCCTTACCCCCAAGACCATGAACTCAGTGAAGGTGAACACTGTTTTGTTATTGATTTGACTCATAGAAAGTAATGAGagagacagacagacagacagacagacagacagacagtgctagctttgcgtgagcagctagtgaggagatgtgttgggacttaacatccccatgactcttgagggctcctcagacattctgcagccacagttctgacactgccatttgggacatgcatatatctgaggccaagactccattgtgcaggaggctcatctttggggctagaatgactgcctgagttgcatgtggccttctcctatcatgccatgtcatctagacccttgctgggggctcttctgtggcatggtggattattaactctgagcctggggagcagggaggtcccatacttggtatgcagagagagaccctgaagagccgtttggcctcacacttcctcccattgggtctccttgccacaggtgtttggcttcttcaagggcatgtccttccccctggccagcatcgccatctataactcggtggtgttcggggtcttcagcaacacccaacggttcctcagcaagtaccgctgtggggaactcgaggctggccctggccgaagtctgtctgatctgctcctggctagcatgctgactggtgtggtctctgttgggctgggcgggcccgtggaactcatcaagatccgactgcagatgcaaacacagccatttcgggaaggtaagaggccgatggagagaaatctggtgtctgaggacccgtgaccctttcccatttgtctggatctgtggtcccattgttcataaccttctctgtgagcaagaccaccctcttccctgctcaccacagggagcatctcattttctcctcttgggagtttcccatgctgcttttgaaggtggctttgacagaggaggaaagagaggagaggggaacccgctgtgtgtcacctttggctgacttgagtgctggaggagacatatttcgattctgtgggttttgttcttggttcagtctcaagtctccatgtctatagaatggaggggctggatctctggtctttcctgtcatagTGTATGTATTATGTATGATGAGTCCCCCCAACCACCCCGCCCTTGCTGGCAGCCTTCAGAATGGTCACTGTGTGAACCCTCTGCACTCTAAGGCTGGGGTTCAGCTGTGGAACATGACAGGAGGCCCACAAGCTCACTCAGGAGCATCCTGGCC
This mutation is a 1016 bp deletion beginning at Chromosome 13 position 56,450,617 bp and ending after 56,451,632 bp (GRCm38/mm10).
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
45651 | CCC CAA GAC CAT GAA CTC AG | Common | A | |||
45652 | TCC CAA CAC ATC TCC TCA CT | Wild type Reverse | A | |||
45653 | TTC ACA CAG TGA CCA TTC TGA AG | Mutant Reverse | A | |||
45654 | Fluorophore-1 | ACA GAC AGT GCT AGC TTT GCG | Quencher-1 | WT Probe | ||
45655 | Fluorophore-2 | AGT GTA TGT ATT ATG TAT GAT GAG TCC CC | Quencher-2 | MUT Probe |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
45651 | 0.40 uM |
45652 | 0.40 uM |
45653 | 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 |