Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.
>chr11:62779690-62779821 132bp TTCGTCAGCTCTTATTCAACACC GGCTGCTCTTCAGTGTGAGT
Mut= 140 bp
Wt= 132 bp
Fam=Mut
Hex=Wt
Wt Sequence (deletion in lower case):
TGCGTTATATTTCTGATCCCCTTTCTCCCCTCCGATTCTCCTGGATAAAGCTATTCGTCTTCATATTCATCCCCCTCGATTTCCTTCTTATTTTACAAACCATCCAGTAAGAGCGTCAGATCTCCTAACTCCACAAAGGCATGCTTCCATCTTGATTCATCTTCACCTGTTATAGGACTGTGTTACTGTTGAGCACAGAAAACAAAGAACATGTGCATTTGCAATGTCTTTTCAGACATGGAAACcagacctgagagcaaggagtcaacttcagtgctagacttttccaaagaagagtcaaacagactgacagaaaatactgcttctgactccgacattgaaactgctcttgaatgtgagcgttggctagaacatcgtcaaagaaatcgggagaggtacccaagacaaatgtctgctcacaggaactccctccctgcgcaagaagatggtgaacgtgatgtatattggaaaagcttcagtcagaaacctctccttacaactgatgacagagaccccaaaggatcctgggacgttcacacgcttgggaaaagactgaagcagaaatcaaagttaatgacaaagcagagaccttataaagagaaaaaacctcataagtgtgatgagtgtggcgaactcttcacttaccgttcggtacttatccgacaccagagagttcacactggagagaaaccttatacctgcagtgagtgcgggaagtccttcagccacagagccaatctgactaagcaccagagaacccacactaggattctcttcgagtgtggcgagtgcaagaaaaccttcatagaaagttcatcccttgaggtacatcagagaatccacatcggggagagaccttacgaatgcgccgagtgtgggaaaggcttcaaccgaagtacacaccttgcccagcatcagctgattcatactggggtgaagccttatgaatgtaacgaatgtgacaaagctttcattcactcatcagctctcattaagcatcaaagaactcatactggagagaagccctataaatgtcaagactgtgggaaagccttcagccactgctcatctcttacgaagcaccagagggttcacacgggggaaaggccatatgagtgcagtgagtgtgggaagactttcagtcagagcacgcaccttgttcagcaccagagaatccacactggagaaaagccctacgaatgtaacgaatgtgggaaaacattcagccggagctcaaattttgcaaagcatcaaagaattcacatgggaaagaaaccctataaatgtggagaatgtggcaaagccttcgttcattcgtcagctcttattcaacaccagagaactcacactggggagaagcctttcaggtgtaccgagtgtgggaaaagcttcaagagcagcccctccctcatcCGACATCAAAGAACTCACACTGAAGAGCAGCCCTGAGAGGTGCTGAGTGTTAGGTGAAGGTTGGCTTTATTGCTGCGTTTCATATTTGATTGTTGAAATAGAGAGACCATAATATGACCCTGGGGTGTGTCTGTGTGTGTATGATTTCACTTGCCCTGTGTATAACTCTGAACAGCAGAAAGGAGATCCCTTCTACCCATTGGCTTGATAATAGTGAAGCCCTCT
This mutation is a 1168 bp deletion beginning at Chromosome 11 position 62,779,722 bp and ending after 62,780,889 bp (GRCm38/mm10).
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
52659 | TTC GTC AGC TCT TAT TCA ACA CC | Wild type Forward | A | |||
52660 | GGC TGC TCT TCA GTG TGA GT | Common | A | |||
52661 | GGC ATG CTT CCA TCT TGA TTC | Mutant Forward | A | |||
52662 | Fluorophore-1 | AGC CTT TCA GGT GTA CCG AGT GT | Quencher-1 | WT Probe | ||
52663 | Fluorophore-2 | CTT TTC AGA CAT GGA AAC CGA CAT C | Quencher-2 | MUT Probe |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
52659 | 0.40 uM |
52660 | 0.40 uM |
52661 | 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 |