Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.
>chr11:90683173-90683261 89bp CACAAAGAAATCCAACTTTCCT AAGAATGTTAATTGTAGGGGTGAA
Mut= 85 bp
Wt= 89 bp
Fam=Mut
Hex=Wt
Wt Sequence (deletion in lower case):
AAGAATCTTAAAGGGATCACTTTTCTCTTGTTGTTTTAGGGTTACATTTCCTGTAACCCGATTCTGTGTGTGTATAGAAGCCAGTGTGTACACACTTCATGACTGTTTATGTAATCCTCTTGCACACATATCTTAGCTGTGTCTCCCCAGGGGGCAGGTGGTGTGATCTATCAATtttggaaggcagcttagagatggtgcgctaagtggcccagctaagacagaaacacaatcaaacattggcctgtacgcttaagccattcctatttccgtattgtacactgcttccatagagactgcctctctatccaaattgttcatttcagtgtctttattgccataggccaaaagatgcagtaaaagctttgaagaaaaggatctccaaaaactataaccacaaagaaatccaactttccttatcagtaagtacttttaatgctataaataagattgcgggggTTTCACCCCTACAATTAACATTCTTTTTTGTTTGTTTGTTTTTGTTTGTTTGTTTTGTTTTGTTTTTGCTTTTTGTTTTTCAAGACAGGGTTTCTCTGTATAGCCCTGGCTGTCCTGGAACTCACTTTGTAGACCAGGCTGGCCTCGAACTCAGAAATCCGCCTGCCTCTGCCTCCCGAGTGCTGGGATT
This mutation is a 284 bp deletion beginning at Chromosome 11 position 90,683,198 bp and ending after 90,683,481 bp (GRCm38/mm10).
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
46100 | CAC AAA GAA ATC CAA CTT TCC T | Wild type Forward | A | |||
46101 | AAG AAT GTT AAT TGT AGG GGT GAA | Common | A | |||
46102 | CCT CTT GCA CAC ATA TCT TAG CTG T | Mutant Forward | A | |||
46103 | Fluorophore-1 | ATG CTA TAA ATA AGA TTG CGG GG | Quencher-1 | WT Probe | ||
46104 | Fluorophore-2 | AGG GGG CAG GTG GTG T | Quencher-2 | MUT Probe |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
46100 | 0.40 uM |
46101 | 0.40 uM |
46102 | 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 |