Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.
>chr3:153850467+153850562 96bp ACACAGCAGCGACACACAG AGGAAAGAAAACTGCCACCA
Mut= 113 bp
Wt= 93 bp
Fam=Mut
Hex=Wt
Wt Sequence: (deleted region in lower case)
AGTTCACACAGATATAGGCACACGTAACTAAAAAAAATCAAATCTTTAAAAAATAAGCAATAAGAAAACTGTTTTTATTTTTAATTTTCATGTTATATTTGCAGAGCATGGTAAGGTTTATGCCCCCACCCGTGGGGGAGACACAGCAGCGACACACAGCTGCAGCGACTAAGTGCTTTGCCCATGTGGTGTGATGTgaaaggcggcttcagcatttggtggcagttttctttcctccttcagtttggtccacagttacaatgtcaaatccttcttaagacattacaggtgaacgttagttacatggtggtcactctgcttttggcttgcagaaccttcacacctgtgtactgccccagccccctgagtggcatcacacctctactctacgtggctcagacaagacagtcaaatatcttaaaaattctcctgcagtatggaatcctagaaagagaaaaaaaccctatcaacattgttctgacaatactactttacccttcgagagtgagaataatggttgaccacgagttgattgacattcaagaagatgccaagacatgtttaatgctatgttccagagtgctttctacgatctcagtcagggagatagaggtaagcggagcaccgcccttctcagggcgtggggagtggataataagagaagcagaggaacacttttggggtttaagcagagctatattctatggggaaaagccgagtgttgctctttggggtgcaatttaccatcagtaagtgttaggacataattgcaaggGAAAATTGCAGAGTATTTTTAATTTATGCATTATTTCCAATGGTGAATTAATGACCTCGCATATGCTCAGTAATTTAAAATGAGGCAAAATTCAAATTAACTAAAATTAGTCCTTCAAATGTTGATTTTTTTTGTTACCGTGAACTTCAAAATGGCAAAGCTGAGTGTCCTATAGACA
This mutation is a 578 bp deletion beginning at Chromosome 3 positive strand position 153,850,524 bp and ending after 153,851,101 bp (GRCm38/mm10).
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
38437 | ACA CAG CAG CGA CAC ACA G | Common | A | |||
38438 | AGG AAA GAA AAC TGC CAC CA | Wild type Reverse | A | |||
38439 | GGT CAT TAA TTC ACC ATT GGA | Mutant Reverse | A | |||
38440 | Fluorophore-1 | AAA GGC GGC TTC AGC ATT | Quencher-1 | WT Probe | ||
39176 | Fluorophore-2 | TGT GAT GTG AAA ATT GCA GAG TAT T | Quencher-2 | MUT Probe |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
38437 | 0.40 uM |
38438 | 0.40 uM |
38439 | 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 |