Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.
>chr5:97456904-97457011 108bp GCATCACCAGGGAAACAGTC AAGCGAGTGGAGTTGGTTC
Mut= 100 bp
Wt= 108 bp
Fam=Mut
Hex=Wt
Wt Sequence (deletion in lower case):
GGACTTAGGTGAGGGGGTAAATAGGCTCCCGAAGCTGTGGGACTGGCAGCTGCGCCTGCGCACTCCACCGTACCTGACTGGCTGTTGTGTCTCCTAACTGGCATCACCAGGGAAACAGTCCACTGACCAGTTTCATGGCAGCCTCgaagcaaacctctgcgggaccgttggtaggagcggtggtccagggaaccaactccactcgctttctagttttcaatagcaaaacagcagaactagtttgctctcatcaggtggaactgacacaagaatacccaaaagaaggatgggtggagcaagaccccaaggaaatccttaaatctgtctatgagtgcatagcgaaagcttgtgaaaaacttgctgaagtgaacattgatatttccaacattaaagctatcggcgtgagtaaccagcgggaaaccactgtggtctgggacaagttcacgggagaccctctgtacaatgccgtggtgtggcttgatctcagaacccagtctactgtggagactctcactaagaaaatcccaggaaatagcaactttgtcaagtctaagaccggccttcctcttagcacttacttcagtgctgtgaagctgcggtggatgctggacaacctaagacccattcaaaaggctgttgaggaaggcagagccatgtttggaaccatcgactcttggctcatctggtgtatgaccggaggagtcaatggaggcatccattgcaccgatgtaacaaatgcatgcagaacgatgctttttaacatccactctttggaatgggataaagatctctgtgacttttttgaaattccaatgagcatcctcccaaatgtatgcagttcttctgagatctatggcctgatgacatcaggggccttggaaggcgtgccaatatctggatgtttgggggatcagtctgctgctttagtgggacaaatgtgtttccatgaaggtcaagccaaaaacacatacggaacaggctgttttttactatgtaatacaggccagaaatgtgtgttttctgaacatggccttctgaccacattggcttacaaactaggcaaaaataagccagttttttatgccttagaaggttctgttgctatagctggtgctgttattcgctggctaagagacaatttcgaaattataacaacctcaggggaagttgaaaatcttgcaagagaggtaggcacttcctatggctgctactttgtcccagccttttcggggctgtatgcaccttactgggaacccagtgcaagagggataatctgtggtctcactcagttcaccaacaaatgccatattgcctttgctgcgttagaagctgtttgtttccaaacccgagagattttggatgccatgaatcgcgactgtggaattccactcagtcatttgcaggtcgatggaggaatgaccaacaacaggattcttatgcagctacaggcagatattctgcacattccggtagttaagtctgtcatgcctgaaacgacggccctgggagctgccatggcagccggagctgcagaaggggtaaatgtttggagtcttgaacctgaagatttgtctacgatcttgatggaacgatatgaaccacaaatccaagccacagagagtgaaattcgtttttcaacatggaagagagcagtgatgaagtcaatgggttgggttacagcaaaggaccctgaaaatggcgataaccctgtcttctcttgtctgcccttgggtttttttatagtgagtagcatgacgttattaattgGAGCAAGATGTGTCTCAACTGCTGAAGAGTAATTTGAACCACGGACTTGCAATGTGTGAACTTTTTACAGAAAGAATTCCATCTTTTAGTGTGAAACCAATATTATAGGTTTATTTTTACCTATAGGCCACTTGCTTACAGGAACC
This mutation is a 1622 bp deletion beginning at Chromosome 5 position 97,455,345 bp and ending after 97,456,966 bp (GRCm38/mm10).
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
42919 | GCA TCA CCA GGG AAA CAG TC | Common | A | |||
42920 | AAG CGA GTG GAG TTG GTT C | Wild type Reverse | A | |||
42921 | ACA TTG CAA GTC CGT GGT TC | Mutant Reverse | A | |||
42922 | Fluorophore-1 | ACC GTT GGT AGG AGC GGT | Quencher-1 | WT Probe | ||
42923 | Fluorophore-2 | AGC CTC GAG CAA GAT GTG TC | Quencher-2 | MUT Probe |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
42919 | 0.40 uM |
42920 | 0.40 uM |
42921 | 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 |