Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.
>chr9:37607544-37607682 139bp AGCTTTGATCCAGCAGAATG TTGGGGAGACCATAACAATTTC
Mut= 132 bp
Wt= 139 bp
Fam=Mut
Hex=Wt
Wt Sequence (deletion in lower case):
CATCTTACTTTTGGAGTTAGTAATTTTTCATGGTGGAAAGTTAAAGAAAACTATCTTTGGATAGAAATATTGCTTGTCTATATGAACTGTTTATATAATGTGTCTCTTTTGAAAACTCTCCCTTCTTTTTCACTTTTCCAAACTTCCGGATCCCTTAGTATATATTTTATATACTTTTTTACATTTGCCAGTGCACATTTAAGTCCATTTATTTAAAATACTGACATTTGTTCTGTGTCTTCCACTTTCTTATGAGTTTTATGAACACAGAAGTTATGAAGGTGTTTGGTGCTTTTGTAAATAAAACTAAGGGCTCATTCAGTGTTAGACTGATCATCATATTTTAATAATTACTTATGTCTACAGcactaggctatttagtgtcagcaagattttcaactgtcagtaaagaaactctgatttgaaacaagaaacttgtgtttctgcattgttcttggttcactttaatgaacacaaaataagtataaaattatttttgttatttagaaaccagctttgatccagcagaatggggggctaaggtagaggaccgcttctataacaaccacgcattcaaggtatagtcttatgaacctaccagatttagCTATATTTTTCTATCTATAAAAGAAATTGTTATGGTCTCCCCAAGTTTTGAGAAGTGTGTCAAGCATTAAATAACTTATTAAAAATTTTTATGATGAGAAATACTCTTCTAGAATATATGAATTGAGTACCAATACTTAGCATTAATTTAAAAATGACAACAGGAGAAAGGACTTGATCATCACGCAATTAGTCCCATCTCTTATTCTTTAAACTTGGGTATCATAAGTCATAAATTATATCTCACAAAATAGAACGCTATTAGTCCAATTTTTTTCATTTGCATGTTACAAAAAGACTAATTCAAATAGAAGATAATGATAGGGATTTTTTTTCTTCAAAGTTAATCTCTTTAGTAACTGA
This mutation is a 241 bp deletion beginning at Chromosome 9 position 37,607,588 bp and ending after 37,607,828 bp (GRCm38/mm10).
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
48249 | AGC TTT GAT CCA GCA GAA TG | Wild type Forward | A | Wt F | ||
48250 | TTG GGG AGA CCA TAA CAA TTT C | Common | A | Common | ||
48251 | AAG GTG TTT GGT GCT TTT GT | Mutant Forward | A | Mut F | ||
48252 | Fluorophore-1 | AGG TAG AGG ACC GCT TCT ATA ACA AC | Quencher-1 | WT Probe | ||
48253 | Fluorophore-2 | AGG GCT CAT TCA GTG TTA GAC TGA | Quencher-2 | MUT Probe |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
48249 | 0.40 uM |
48250 | 0.40 uM |
48251 | 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 |