Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.
>chr16:30356912-30357023 112bp GTCTCGTCTCCATCGTACCC ACATGCTTCCTCTCAGAACACA
Mut= 109 bp
Wt= 112 bp
Fam=Mut
Hex=Wt
Wt Sequence (deletion in lower case):
AAATAGGAGTCTAAAGGAAGTAAACCGCTAAAGTTTGATTACCACTATTGATATCTTTGATTTAAATAGTGTAGCTAGCTAGCAATTTTATTTTACTTCATTTATTTATTTTGGTTTTTGAGATAGGATTTCTCTGTGTATCCTTGGCTGTCCTAGAACTCAGTCTGTAGACACAGTTGGCCTCGAACTCAGATCCAGCTGCCTCTCTCTCCTGAGTGCTGAGATTAAATGTGTGAGCCGCCATGACCTAGCAGCTTTTTTATTTTTAAAAGTAGAGAATTTTATAGTCATGgaataagacttggaagcatgaatctcagaatggttgactttagccatatctaacataattcttcttctaaattattaatataattactgcatttttctgtcatagaaagttgatttatggagtaaatgaaattgctgtgaaagtgccttctgtttttaagcttctaattaaagaggtaagactttgtatataacccaacaaacagtagagtcatccatttggaaagagtcatgtttaagaagctgttcttagtttcctgctttgcttcatattttacttggaaggtaaaaattgaaaaattcttgtctagtagaaaaggtatgagtatgcattttttattatatagatttctttgtgtctcgtctccatcgtaccctggttaagtgtttgtgacttgctcccttgctcctttacttcacattgGCTAGTACTGGAAACTGAACATTTGTGTTCTGAGAGGAAGCATGTTAGTTCTTGCAGTGAGGATTCATTTATAATTGCCTGGGCTTTCTGAATAAAAGATTAGGAATGTGTATGGGAAGTTGGTGAGTAACTGCCCAGCAGAAAGACTTGCCAGTCTTTCTTTGGGTTTTGGCTATAGATAAAAAGTAGTATTGGGCGGATACTTTACTAGAAACAATGATGTTTTCCCACCACTTTCTTAGAGCAGCCCTTTGGTGCTGCCTGAGAGAATAGTTTCAGGTATGATTAAGACA
This mutation is a 423 bp deletion beginning at Chromosome 16 position 30,356,957 bp and ending after 30,357,379 bp (GRCm38/mm10).
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
48044 | GTC TCG TCT CCA TCG TAC CC | Wild type Forward | A | |||
48045 | ACA TGC TTC CTC TCA GAA CAC A | Common | A | |||
48046 | ATG TGT GAG CCG CCA TGA | Mutant Forward | A | |||
48047 | Fluorophore-1 | ACT TGC TCC CTT GCT CCT TTA | Quencher-1 | WT Probe | ||
48048 | Fluorophore-2 | AGA ATT TTA TAG TCA TGG CTA GTA CTG GA | Quencher-2 | MUT Probe |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
48044 | 0.40 uM |
48045 | 0.40 uM |
48046 | 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 |