Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.
Mut= 85 bp
Wt= 100 bp
Fam=Mut
Hex=Wt
Wt Sequence: (deleted region in lower case)
CATTTTTTATTGTTTTGACATTCTCTAGGTAATAAATGTTATCAGTTTAGTAGACTTACTTTTTTGTTATATTATGTGATTCCTCTTGTCATTTGTCTTATATTTTTTAACTGCTGAGCCTTTGACTATGGTAAATATTTTTAGTGGCATAGTGGCTAATCATTGATATTGCGAGAGGATGTCTGCTTTGCTTTAGGAGTATAGATTATTCCTAAGTCACTAAGTTGATTGGCATTTGATTGACTCCTAACTTTGTGAAGACACTTTAATGTTATGAAAAGATGTGGAAGAGAGAAAATTTCTTATATTTGAGAAATAGTACAACAGATATCTTTATAATCTGTTGACTGGCAGGATCACTATAAAATAATACTTGCAGAATACTCCATCCTTTAATTCTAAAGCAGACTTATTGCCCTGGAAGGGCTTGTCTTTTTCAAGAGATGGTCTCAGCGTCATAAGGTATTTGCACATTGGAAGGCCACTGGGTTCTCTTCTGACCAGCAGGTTATATCCTgtggggagccagtgtttgggcttgtgaatgagccatatatttttctcccctctatggcatttctgacagcagagctttggagtattagttttaaatggacgaaatgataaacctactcatgtcatgcggactctgatttgtgttccgtttttccccagatgccctcattgcgcaaaagagtgaaaatacaggccgagaatgcagaacccaaagatgaatgctctttcttctctgagttgtaagcatattagtgtaatatgttgttgggggaatggtgactattttaagagtaatGTGGTCTGTCCCGTAGACACTTAAGTCATTTGGGTCGAAGACATGTGTGTTCTTGTCCTTGCTCTATGTAACAACTCTCTGTTATAATTGTGCCATGATCCAAGAGAGAGAAAGCAGCTATCAGGTTATGTAATGTTTACCCTGTACAGGTGAAAGAGGGCAAGCTGTTGTTTACACTGCTTTGGAGCAGAGCTCTTTCTTTTTTATGATATTTTTCATGTGCATTGGTGTTTTGTCTGAATATCTGTGTAAGTGTGTTGGATCCTGAGGAACTTGAGTTACAGATGGTTGTGAGCTGTCATGTGGGTGCTGGGAATTGAACCCAGGTCCCGGAAGAGCAGCTAGTGTTCTTAACTGCTGAGCTATCTCTACAGCCCCTAGTTGTAGCTCCTAACCCATTTAGCATTGCTACTGTGTATAGGGTCACCTTTTGTTTTGGGAGCTGCCTGATGCTTTATAGGACATTTAGTGGCATCTGTGGCCTCTACTAAATGTGGCAACAGTTCTTGATATGACAACCAAAAATGTCTTTAGATAATGCCAAATGTCTCAGAGGCTCAAAATCATCCTCTGCT
This mutation is a 294 bp deletion beginning at Chromosome 17 negative strand position 86,142,597 bp and ending after 86,142,304 bp (GRCm38/mm10).
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
37360 | GCC ACT GGG TTC TCT TCT GA | Common | A | |||
37361 | GAA ATG CCA TAG AGG GGA GA | Wild type Reverse | A | |||
37362 | ACA CAT GTC TTC GAC CCA AAT G | Mutant Reverse | A | |||
37363 | Fluorophore-1 | AGT GTT TGG GCT TGT GAA TGA | Quencher-1 | WT Probe | ||
37364 | Fluorophore-2 | TGG TCT GTC CCG TAG ACA CTT A | Quencher-2 | MUT Probe |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
37360 | 0.40 uM |
37361 | 0.40 uM |
37362 | 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 |