Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.
Mutant= 111 bp
Wild Type = 136 bp
>chr12:104344449+104344584 136bp AGGGCCTAGAGTTGCTCCA TGTATTTTGGGTGCCAATGA
Sequence (deletion in lower case)
AGTATATCAGAGACTGAAACACAAATGTACCCTTTGGGCTCCTCTTAGCATTGAGGAAGAAAGAGTGTTGCAATGAAAATTAAATTAACCATCATCAAATAGGTTAAAATGTGAGAAAGGGTAAAGTTAAGAAGGGGAGTGTCCAAAGATTAGGAAAGCTGGAATCTGATGCAGTGTGCGATTTTAAATAGAAGGCTCATTAGAAGATGTTTTAGGGTTGCCATGCTAGGGGAGAATTGTACTGTGGATGTAGGAGGAAAGCATACTAAGTCACAGAACGGCTAGGAACAAGTCAGGTTGCCTGTCCACTGTaaacactctcgggacctgttggatgattaatgcattagaaagagcaaagtcaagtcctctaggcatggataggaagaggattttctccagggagaaggctgtgtggtgaggagacccagcgacatgatggattgagggggggtcatagagtccagggactcttaccttgctcttttttattctcagccaaatagaggagctaaacctgcccaagttctccatcgctagtgactacaggctggaagaggatgtccttccagaaatggggattaaggaagtcttcacagaacaagctgacctatctgggatcacagaagccaagaaactgagtgtgtctcaggtaatacaagaagcacaggatgttttgccttgggtcctgaatgggggtggtgaagagcatgtaggctgtggggatggaacatgtgtgagaactacattgctgaggttccttaacaaatgtccatcttctacccctgtacataagctcacctaggagcccatagtggagagggcctagagttgctccatagaaccatggatactaacatgtgctgccctgcagggcaacatacagtacactgcaggggcacctacaatacactaCAGGGGCACCCACCATTCACTTCATTGGCACCCAAAATACATTGTAGGCCAAACCACTTCCATACTTTACCTCCTATACTACAGTGCTGGGACAGTACTTCTTGACCAGCACTCAGATTATTAATAGTAAAAGTAATGACAAGGAGTCATGGTGGTTTTGAGGAAAACTCCAAGAATGGGAAAGAAGATGCTTTCGACCCCTATACTACATTCTTAGGTGCACAGCCTGGCTGTAACCAATGTTCAGTCCAGCGCCCACTCAGAGCTCTGGGTACAGATCAGTCCCAGCAGGACATGGTATTTTGAGAATGTGAAACCATGGGTTGCA
603 bp deletion beginning at Chromosome 12 positive strand position 104,343,941 bp and ending after 104,344,543 bp (GRCm38/mm10).
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
37781 | TGT GGA TGT AGG AGG AAA GCA | Mutant Forward | A | |||
37782 | TGT ATT TTG GGT GCC AAT GA | Common | A | |||
37783 | AGG GCC TAG AGT TGC TCC A | Wild type Forward | A | |||
37784 | Fluorophore-1 | CGG CTA GGA ACA AGT CAG GT | Quencher-1 | MUT Probe | ||
37786 | Fluorophore-2 | CTA ACA TGT GCT GCC CTG C | Quencher-2 | WT Probe |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
37781 | 0.40 uM |
37782 | 0.40 uM |
37783 | 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 |