Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.
>chr16:20654328+20654457 130bp GGCAGAAGCATCTTTCTTTGA AAAGTTCCATGCTAAGCCTCA
Mut= 133 bp
Wt= 130 bp
Fam=Mut
Hex=Wt
Wt Sequence (deletion in lower case):
TTTTTTTTTTTTTTTTTTTTTTGGTTTTTCGAGACAGAGTTTCTCTGTATAACTCTGGCTGTCCTAGAACTCATCTGTAGACCAGGCTGGCCTCGGACTCAGAAATCCACCTGCCTCTGCCTCCCGAGTGCTGGGATTAAAGGCGTGCGCCACCACTGCCCGGCAGAAGCATCTTTCTTTGATTCCCCcattgtttattactcttgagttaggttagcttcagagggagctagcaacaccaactttagggtttttttttttttttttttttgaggcttagcatggaacttttctgtatagagactagtatatattgaattttataggtgaattttgtggacagtaatcttgattctgtaatggcacatgctgttttcctagtgttttgcagctgacatcatctctgttttggccatgaccatgagtggtgagcgggagtgcctcaagtatcggctcgtgggctcccaggaagaactggcgtcatggggccatgaatacgtcaggtaaggcattttctttatggacatgagaagggacttggggtcattattctgtttgtttgggaaactgaatgtattgaatcccccagacactgaattccttgactcccagcaacatgcttccctaatgagcaagtctatctatccatagacatctggcaggagaggtggccaaggagtggcaggagctggatgatgcagagaaagcacagcgggaaccactgcttaccttggtaaaggaaatcgtcccctacaacatggctcacaatgcagagcacgaggcttgtgacctgctcatggaaattgagcaggtggacatgctagagaaggacattgatgagaacgcctacgccaaggtctgcctctatctcaccaggtgagtgagtacaggagagcaggtagcactcgtcttccagggagtcttagggttcctcttgaaggaagagactttgtgactccttagtaatcctagaacatgctacattgccttctgtatagtaggcattcagtgtgtatacttgggaagggatttaaataagttacatgagttgcacgtcctgaaattgtactacttttgactttgtagttgtgtgaattatgtaccagaacctgagaactctgccctcctgcgctgcgccttgggcgtgttccgaaagtttagccgtttccctgaagctctgagattggcactgatgctcaatgacatggaactggtagaagacattttcacttcctgcaaagatgtgtatgtagagaaattggcaaaaggaaagatgagtctgtgtatgtgctttgtgtaaaaatcagagacgtagcagtttctgatgcgacacagaggcttttctgtctcatagtggctaagagtgagcaggtgtgaccgcttcccccacagggtggtccagaagcagatggcattcatgctgggtcggcatggagtgttcctggagctgagtgaagatgtagaggaatatgaagacctcacagaaatcatgtccaatgtccagctcaacagcaacttcttggctttagctcgggaggtgagggtctcctcttggtcctgaagggttttggccgaagttctgtcattctacctccctgactaatacctttcaatagactccctggttaaagttgctattcagtgggagaacagaagggacagctaggtaggggagtactggggaagcagttgtgaccctctggctttttttcagctggacatcatggagcccaaggtgcctgatgacatctacaaaacccacctagagaataacagtgagtagctctgtgggcattggagggactgttaattgttctaattgtattggGAATAACAGCTTAGACAACCCATTTCTAAAGCACTTCAAATTCTTATGGAGACATAGATTAATTTAAAGTTGGAGACTGGGCTAGCAAGATGGCTCAGTGGTTAAGAGCGCCGACTGCTCTTCCAAAGATCCCGAGTTCAAATTCCAGCAACCACATGGTGGCTCACAACCATCCGTAACGAAATCTGATGCCCTCTT
This mutation is a 1640 bp deletion beginning at Chromosome 16 position 20,654,355 bp and ending after 20,655,994 bp (GRCm38/mm10).
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
40779 | GGC AGA AGC ATC TTT CTT TGA | Common | A | |||
40780 | AAA GTT CCA TGC TAA GCC TCA | Wild type Reverse | A | |||
40781 | CTT AAC CAC TGA GCC ATC TTG C | Mutant Reverse | A | |||
40782 | Fluorophore-1 | CAG AGG GAG CTA GCA ACA CC | Quencher-1 | WT Probe | ||
40783 | Fluorophore-2 | CCC CGA ATA ACA GCT TAG ACA A | Quencher-2 | MUT Probe |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
40779 | 0.40 uM |
40780 | 0.40 uM |
40781 | 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 |