Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.
>chrX:73905847-73905946 100bp ATCTTGGGGGCGCTAAGT TGGTACATCTTCTTGGTCTGC
Mut= 93 bp
Wt= 100 bp
Fam=Mut
Hex=Wt
Wt Sequence (deletion in lower case):
GGCCATGGCTCTTACTATGGAACAGGTTTGACCTGGCCTGAAGTCTCTCCAGCTTGGTGGAGTACCACAGCAGTCCTGCCCCTGAGCAGCTGCTTGTGTTTGTACAGAAACCACAGTCCAGAGTTCTCTGCCTTAGGGCCCTTCCCAAGtgcacaggggcttctgaacctgagtactttccttcggggcacacttctgttctttgctctagcttttagctcagtcaggtgccagggagcctttggtgtagacaggttgtctgtctctgcatctcctatgtctttctggtctggttggttcacggcttgcatgtctgcccgccctaaaccaaggtccccttgcagagatgcgctggcagttgagtgagcagcttcattgcctggagcttcagggagagcttcggcgggatttgctgcaggagctagctgagttcatgcggcgccgggcggaggtagaactggagtattctcggggcctggacaagttggctgaacgctttaccagccgcagtggtcgccttgggggcagcagccaggagcagcaaagcttccggtaggtaccacactagcagtagtgccttgcaggtcctgagacctaggctccttggtctgagttctcctctgctacaggaaggagccgactctcctgtcacccttgcactgctgggctgtgctgctggaacacactcggcagcagagtcgggaaagtgctgccctcagtgaggtgctggctgggcccctggcacagcgcctaagtcacatcagcgaagatgtgggacgtattgtcaaaaaggtagggtctgtggaagggctcagaatctaaccggaaaaaaaaaaaaaagcacatggggatctatctgtggttgagaggcatgggaggccaggtggaaattgtagtccaaggtcgtctcatcccaatctgaggtgggatgagttgtcacaggatgctgggaggcattaagtagagggatgctatggtcacacattccccttcatctctagagcaaggacctggtgcagcagttgcaggctgagctcctggaagtggtctcagaactccagacagtaagcaggacctgctgtctactaacaacaggaagcaggaacagctgctgcaagccattgcatctcccaatcaggctcaaaaaatggatgctcatgggagacctgagtctttagtctggggggcaacaggcatgggcccctctatgctctgagaacagagtagttaaataacaacacacttaggtgaaatggcccaaatttcagatctgaatcttgggggcgctaagtggggctgagggactcttcttagtcatgaccttcaGCAGGGCTACTTTGCCTGGGCCTCCCTGCAGACCAAGAAGATGTACCATGTGTACCACTCAGAGAGCATGAATGCTGAGACTAAACTTCGGGAAGCGGAGCGGCAAGAGGAGAAGCGGTCAGGCAGGAGTGCCTTATCCACC
This mutation is a 1177 bp deletion beginning at Chromosome X position 73,905,895 bp and ending after 73,907,071 bp (GRCm38/mm10).
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
42979 | ATC TTG GGG GCG CTA AGT | Wild type Forward | A | |||
42980 | TGG TAC ATC TTC TTG GTC TGC | Common | A | |||
42981 | CAG AAA CCA CAG TCC AGA GTT C | Mutant Forward | A | |||
42982 | Fluorophore-1 | CTG AGG GAC TCT TCT TAG TCA TGA C | Quencher-1 | WT Probe | ||
42983 | Fluorophore-2 | CTT CCC AAG GCA GGG C | Quencher-2 | MUT Probe |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
42979 | 0.40 uM |
42980 | 0.40 uM |
42981 | 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 |