Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.
>chr3:86906366-86906452 87bp TTCTGGGCTTAGGGGTTTTC CCTTTTCCTTTAACCTTCTCCA
Mut= 90 bp
Wt= 87 bp
Fam=Mut
Hex=Wt
Wt Sequence (deletion in lower case):
GGAAGTCCTTTATAATTTAGTTACTTGCTCTATTTAATTCTGCAGCCAAGGATACTGAGACTAGAAGTGTGACATAATGTCTTATCAAAAGTGACACATTTGGGTAAAATAAGCCCAGATCTCACTCTTAGTTTCTGGGCTTAGGGGTTTTCACATCTCAGAAAAGCAGTctacgtaattgagaagggagggaggtttggagaaggttaaaggaaaaggttgtaatgtcccttttcctgtgaccccccaaacttagggcttggcttgcaggtctctagaagctgttgtttacatataatggattcaggtctactatggccagtgctcagagatctgatattatgctaattattatttttgttttttaggtgaaagttacgtgtgtgcctccaacgaaccatttcgtaaagttgattacaccaaaaacgttaatccgaactggtctgtgaacatcaagggcggaaccacccgcaccctggccgtggcctctgcgaagagtgaggtgaaagaaagcaaagacttcattaaacccaagttagtgactgtgattcgaagcggagtgaagcctagaaaggccgtgcggatccttctgaataaaaagactgcccattcctttgagcaggtcttaacagatatcaccgaagccattaaactagactcaggcgtggtcaagaggctgtgcaccctggatggaaagcaggtaagtgcttctgcctgcagctcgattgttgattatccttgtgggtatttgaagagatgcctggaacttgctgtgtagtccaggctgaccttgaacttgggatcttcctggtcctgccaagtgctaggattagggtgtgcaccatgcccaactattgcagttagacattccacagaatgtcctggtctcctggaagtaatcctagtGGACTTTTTTTATGAAGGATTGTGTGTGTGTGTGTGTGCGTGAATTAGCCTTTAGCATAAAAAATATAAAGGGCTTCCGTTTCTCCAATTATCTAAGAGCCCATTATATGCCTCTTCAGTTTGTAAAAGGAAAAGATTGAATTCCTGGGGAATAAGTTGCAGTACTGTCCTATGCTAAG
This mutation is a 736 bp deletion beginning at Chromosome 3 position 86,905,679 bp and ending after 86,906,414 bp (GRCm38/mm10).
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
44702 | TTC TGG GCT TAG GGG TTT TC | Common | A | |||
44703 | CCT TTT CCT TTA ACC TTC TCC A | Wild type Reverse | A | |||
44704 | AAG GCT AAT TCA CGC ACA CA | Mutant Reverse | A | |||
44705 | Fluorophore-1 | ACG TAA TTG AGA AGG GAG GGA G | Quencher-1 | WT Probe | ||
44706 | Fluorophore-2 | AGC AGT GGA CTT TTT TTA TGA AGG | Quencher-2 | MUT Probe |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
44702 | 0.40 uM |
44703 | 0.40 uM |
44704 | 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 |