Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.
>chr19:5893009-5893101 93bp CATGGAGCTGAACACCAAGA TGGGACATCTTAGACTCATCCA
Mut = 91 bp
Wt = 93 bp
Fam = Mut
Hex = Wt
Wt Sequence (deletions in lower case):
CTTCCCAGGGAGTCATGGAGCTGAACACCAAGAAGAAGCTTCACGCCCTgtccctggctgagaagatccaggtgctggaactcttggatgagtctaagatgtcccagtcagaggtggcccggcgcttccaggtctcacagccccagatctcacggatctgcaagaataaggagaagctgttggcagactggtgcagtggcacagccaaccacgagcggaagcggaagcgtgaatccaaatacagtgggatcgatgaggctctactgtgctggtaccacattgcccgagccaaggcctgggatgtaacggggcccatgttgctccacaaggccaaggaactggccgatatcatgggccaggatttcgtgcctagcattggctggctggtccgctggaaacgtcgaaacaatgtgggctttgggactcgccaggtccttgtccctctgttccctcctgaagctcctcctgcagttctcccatctcaggcccagccacctctttctcttaaagacttctcaccagaagatgtttttggctgtgctgaagtacccttgttgtatcgcgcggtgcctggcagagtgtttgaatgtgatcggttgcaagtactgttatgtgccaacagcagaggcacagaaaagcgaagggtatttgtgggtgggctccaggcggccccaagatgcttctttggggtgagcagtgaggcactgcctacctcttatcaccctgacctagccattccctggtcagagtggctggcacagtttgaccaggacatgggacagcagggccgacaggtagccttgctactggcttctggagtggtagaggaatgggccagccttcctggactccatcacgttcgactcctgcctctctctgcctccagcaccactccttccctgcctggctctgtgattttggcctttaaggcccattaccgtcaccgtctactgagcaaactggctgccatgcagagtgggaaagagggcacctccctggcggaggccagggctagcatcacagttttggatgctctgcacatggtggccgcagcttgggccaaggtacctcgccagctcattttgagcagctttgttcaagaagggctcgctccgggcaaaacaccactgtccctggacaaagacactgagatgtcaccagtccctagtgggctgagccaagaggagttttctcactttgtggacctggaggatgaggacccagggcctagagtgtgcaaagaggagacgggtactgaagacagtgggagggaagaagatggctttgagcccctgcccaccaaagccgatgccctgcaggcactgtgcaccctgaggaggtggcttgaatgcaacagtgcctctccagaactctttgagaaattctatgactGTGAGGTGGAGGTGGAGCAGCTCTGCTGTCTGTGAGGAGGCATAGCTGCCTATTAGTCTCCCTCTCCTGTTTCCCATGGAAACGCCTCTCCAGAGACAGACTGGCTCTTTCCTGTGGAAATAG
This mutation is a 1343 bp deletion beginning at Chromosome 19 position 5,891,723 bp and ending after 5,893,065 bp (GRCm38/mm10).
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
52235 | CAT GGA GCT GAA CAC CAA GA | Common | A | |||
52236 | TGG GAC ATC TTA GAC TCA TCC A | Wild type Reverse | A | |||
52237 | TAA TAG GCA GCT ATG CCT CCT C | Mutant Reverse | A | |||
52238 | Fluorophore-1 | TGG CTG AGA AGA TCC AGG TG | Quencher-1 | WT Probe | ||
52239 | Fluorophore-2 | CCT GTG AGG TGG AGG TGG A | Quencher-2 | MUT Probe |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
52235 | 0.40 uM |
52236 | 0.40 uM |
52237 | 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 |