Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.
>chrX:74371058-74371161 104bp AGGCAGCTGTGCTGAAAGAG TGCAAGCTTAGGAGCATGG
Mut = 101 bp
Wt = 104 bp
Fam = Mut
Hex = Wt
Wt Sequence (deletions in lower case):
GAGGCAGCTGTGCTGAAAGAGGAGATGGCTGAATGACATTCaagggccagcattatttcaaaactagaggtaaaggtctgagagccccatgctcctaagcttgcaatttctcctcttcattaaccactttctttaccccttttcttttcaggaaaaaatcctgctatcaaggaagaaaatataggaccaggaccttcccatggacccttctcaggacattccaacagcacaagcaagggtgtctccctaagacacaagaggcctgaaaaaactatcctttgtgaagtgccatggtggtcaagaaaagcagaggtagctcccagcagttgcctggcctgggaggacagggtggttgtacaggttttgtaggcatgcttggaactgccctgctgttcatcagcctaccatggggagctcaagtgatggccagtgccaacatcagcactgctctgggccacacaagtggtcactatttgagtattggggatggctcagtgacagaatttgatttccctgagaagagtgagggtatcattgtgatctctagccaatacactggacagaccaatgggacaggccccagccccatacttaaggttatatccctggacacagaggtgctgaccattaagaatgtgagtgccataacctggagcagtggaggtggctttgtggtaggcatccactcaggtctagctgggctggccccactccacctccagctcatggatttctatgaggccccacccctgctgatcgaagaacggagagatttctgcatcagggtgtcacctgctgaagacctcccttctgctctcaacaccaacttgggccacttctcagagaacccaatactctacttgcttctgcctcttatctttgtcaacaagtgttcatttggttgcaaagtggaacttgaagttttgaaggagctcctacagagcccccaacccatgctgttgggcctcttgggccagtttctggtcatgcctttctatgctttcctgatggccaaagtcttcatgctacccaaggccttagctctgggcctcatcattacctgctcatcacctggtggtggagggagctacctcttcagtctcctccttggaggagatgtcaccctggccatctccatgactttcatctcaacagtagctgccactggtttcctaccactctcatcagccatctacagctaccttctcagcatccatgaaacactccacgtgcccatctccaagatattggggaccctgctgtttatcgccatccccatagcagcgggtgtggtgatcaaatctaagctccccaagttctctgaactactgttacaggtcatcaagcccttcagctttatacttctcctgggtggcctgttcctggcctaccacatgggggtcttcatcctagtgggagtcaggttacccattgtactggtgggtttcacagtgcctcttgttggcctcttggtgggctacagcctggccatctgcctgaagctgccagtggctcagcgacgaacagtcagcattgaggtaggggtgcaaaacagcctgctagccttagccatgctacagctgtctctgcgccgccttcaagcagactatgcctctcaggcccctttcattgtggcactgagtggtacctccgagatgttggctttggttattggccagttcatctatagcatcttgtttcctgttccctgagccctctggatcaagttttataacttccagcccccatactcctctcttcccacagttcttgtgtaccgtaccaaaggcctttagttctcatgcactatgcactcaagacaaatccaggcttatttttttactccttttcgccccagctttcagtgccaaagaggccatgctgagttagatagttgggtattgcccagaaatatatttcaataaaaaaaaaccaaaaaaacaaaagcaagctcgggtcccttccagtctgtcccttggagcctgtggattggggtaagaagagtgtgtctgggcggatatgggccagaacagtgaaccagcttggtgaccctgcacagaacccactttctcctcctcactctgttttgcacatggtagacatgtacctttgagctgtatcctcagatctacaaaggctttgctctaacagccctacctaacagggcagacctctgcACTATAAGGAGTGAAGAGAGCACTACTTCCGAGCTTCAGTCAGGATATTTTTGCAGCCAGACTAGAAAGGAAGTAGA
This mutation is a 2136 bp deletion beginning at Chromosome X position 74,368,986 bp and ending after 74,371,121 bp (GRCm38/mm10).
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
51320 | AGG CAG CTG TGC TGA AAG AG | Common | A | |||
51321 | TGC AAG CTT AGG AGC ATG G | Wild type Reverse | A | |||
51322 | TCT GGC TGC AAA AAT ATC CTG | Mutant Reverse | A | |||
51323 | Fluorophore-1 | AGG GCC AGC ATT ATT TCA AAA C | Quencher-1 | WT Probe | ||
51324 | Fluorophore-2 | CAT TCA CTA TAA GGA GTG AAG AGA GCA | Quencher-2 | MUT Probe |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
51320 | 0.40 uM |
51321 | 0.40 uM |
51322 | 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 |