Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.
>chr5:30959413-30959501 89bp GCGGGAAGCAAAAGTACAAC CTGTGAGGGAACCAAAGTGG
Mut = 89 bp
Wt = 89 bp
Fam = Mut
Hex = Wt
Wt Sequence (deletions in lower case):
GCTCTAAGCAACAGCAACAACAACCAAAGAAACAAAAAAACAAAAAAAAACAAAAAATAAGAGCTGGAGGTGCGGGAAGCAAAAGTACAACTGAGAGAGACTAGGATTTAATgtgacttccgggctggcctgacctttgcccactttggttccctcacagcattttctgcagctagagctgatcaacgggtgcctgagcgcttccttgctgcactctcatgacacggagacacgggccaccatgaatttggcgcttgctggtgacattcttgctgccggacaggatgcccagtgtcagcttcttcgtttccaggtccatcaacagaagggcagtaaagcggagaagtcaggtgaagagctttggaaaaagtcgctctaattcttggtctccaaaaacgagagattaggtgtggggcagtggtcacattgaagtctgtacttgggtcactagaggactggtgttagttaatacagatgctcaggtccatctgagggaggtggtgcacagctgtagtctcagagcttaggagtggaggcaagaggatatttaaggctagcctggggtgcctagcaaatccctgtcttaaaaaacaacaacaaaggaatctggttcctggcaaattgtttgtgttgcaggttccaaggagcagggacctcgacagagaaagggggctcctccagcagagaagaaatcgggagcacaagttcaccccgaaggggttgaactcaaagtaaagaatttggaggcagtacagacagacttcagcaatgaaccgctgcagaaagttgtgtgcttcaaccatgataacaccctgcttgccactggaggaactgatggtcatgttcgtgtctggaaggtgtggttttaggggcttgggggggtgggtgagtgttagttgcaataacaccaaaattgtgagaatctgaatatgcattctgtgaaacttgagtggccattggggtaagagggatggggatttcagcgttctcgctgtctggtctgtaactgtcttggctgtaggtacctagcctggagaaagttctggagttcaaagcccatgaaggggagattggagatttgactttgggtcctgatggcaaggtaaggaggtggagatgtgggatgctggtgcagaaaggcagagcctattgtgtgccttgcacagtacatagatgtcctaactgtccatctttgggatctttactcctccccagctggttacagtgggctgggactttaaggcctctgtgtggcagaaggatcaactggtgacacagctacagtggcaagagaatggaccagcctcttctaatacaccataccgctaccaggcctgcaggtgtgcaggtcacagggtggctggcaggcacacctcagctgggaaatgggaaaagacagaaggagctggccatggctggagccttgttggaggttgttccaggtgtgaccagggtgcagggcacaaacatgagggctgacaggaactgaaagccttttgcctggcctccaggtttgggcaggttccagatcagctcggtgggctgcgactcttcacagtgcagataccccacaagcgcctacgtcagcccccaccctgctacctcacagcctgggacagttccaccttcttgcctcttcggaccagatcctgtggccatgaggtcatttcctgcctcagtgtcaggtgtgaagtcctactgcttgattggatggtgggcgcttgagggagctggggaatgagtcctgactcagtctctggaggaccactgttGTGGGGAGGGAGTTTGCACAAGCTTCAGAGCAATGAGCCCTCTGTTGTGTGTTACAGTGAACCCTCTGTTGTTGTATGTATGTTACAGTGAATCCTCTGTTGTATGTTACAGTGAATCCTCTGTTGTATGTATGTTACATTGAATCCTCTGTTGTGTGTTACAGTGAATCCTCTGTTGTGTTAC
This mutation is a 1660 bp deletion beginning at Chromosome 5 position 30,957,801 bp and ending after 30,959,460 bp (GRCm38/mm10).
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
51203 | GCG GGA AGC AAA AGT ACA AC | Common | A | |||
51204 | CTG TGA GGG AAC CAA AGT GG | Wild type Reverse | A | |||
51205 | ACA ACA GAG GGC TCA TTG CTC | Mutant Reverse | A | |||
51206 | Fluorophore-1 | ACT TCC GGG CTG GCC T | Quencher-1 | WT Probe | ||
51207 | Fluorophore-2 | ATG TGG GGA GGG AGT TTG C | Quencher-2 | MUT Probe |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
51203 | 0.40 uM |
51204 | 0.40 uM |
51205 | 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 |