Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.
>chr5:65375590+65375751 162bp ACTTTTGTAATGTGCTGTGTCC CCTGCCACTCAAAGCCATC
Mut= 164 bp
Wt= 162 bp
Fam=Mut
Hex=Wt
Wt Sequence (deletion in lower case):
ACTTGGTCCCCTTGTACCATGCAGGCCTCTGACTCAACTTTGTCAAGCTTCATAGTAGGTGCCTTTACAGATGCAGCTATTTTGCTGGCCCCAAATCAATAATTTGTGGTTTGATATAAAATGCTTGATATTTAGGAAAAGAGTTACTTTTGTAATGTGCTGTGTCCTGgcactttgtaaacgaactagaatgttttagtcctctgaaaatcgctgcttccttttctctgtgcagcaataaaatttgatgaaatccgcgtgtttggttatacggcctggactctcctggatggctttgagtggcaggatgcctatacgacccgacgagggctgttttatgtggactttaacagtgagcagaaagagaggaaacccaagtcctcggctcattactacaagcagatcatacaagacaacggcttccctttgaaagagtccacgccagacatgaagggtcggttcccctgtgatttctcttggggagtcactgagtctgttcttaaggtaagtggctacagctgaattaaccacgcaccagGGGAGATAGTATACAGTATGGTTTTTTGTTTTGTTTTGTTTTACAGTATGTTTTTACAAGTGTAAAATACATGTAAGGATATCTTAGTCATTGTTCTGTTGCTGTGAAGAAACATCATGACCACAGCAAGTCTGGGGTCTTCAATTGGGGCTGGTTCACAACTCATCAGCATGGTGAGAA
This mutation is a 369 bp deletion beginning at Chromosome 5 position 65,375,614 bp and ending after 65,375,982 bp (GRCm38/mm10).
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
46801 | ACT TTT GTA ATG TGC TGT GTC C | Common | A | |||
46802 | CCT GCC ACT CAA AGC CAT C | Wild type Reverse | A | |||
46803 | AGA CCC CAG ACT TGC TGT G | Mutant Reverse | A | |||
46804 | Fluorophore-1 | AGT CCT CTG AAA ATC GCT GCT TC | Quencher-1 | WT Probe | ||
46805 | Fluorophore-2 | AGT CAT TGT TCT GTT GCT GTG AAG AAA | Quencher-2 | MUT Probe |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
46801 | 0.40 uM |
46802 | 0.40 uM |
46803 | 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 |