Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.
>chr1:74280664-74280768 105bp CTGGCAGCTCTAGAAAGTGG ACAGGACACAGTCAGGCACT
Mut= 102 bp
Wt= 105 bp
Fam=Mut
Hex=Wt
Wt Sequence (deletion in lower case):
GGCTGGCTGAGATGAGCATGGTGCTGGGGTAGGAGTAGACAGAGGCACAGAGAAGATAGGCTCTGGTTGCTTAACAGTGGGGCCAGTAGTATCTGTTCTTGGGAAACGGGAGTGCTAGTCCCTGGagctgcagcaagtttggcccccttctttgctgaggtcctcctactttcttttctgctcagttgcctttaaacagtgactagatgcatgggtcacttgtccccaacatttaggtattttttaatgttcttttttgccacctttttaaaggttatatagttgggttggccttggatgttggcaaggctggcctcaaacttggtacatctcttaggcaaacttgagtttaaaacctgcctacttctctttgctgcaggccacaaagactctgtgacatgcgctggtttcagtcacgattccaccctagtggccacaggggacatgagtggtcttttgaaagtatggcaggtagacaccaaggaagaagtctggtcctttgaagcaggagacctggaggtgagatccttggagtgagcttccactctttgggaccttaagtgagagggatagcggcagacggatccagatctagcctggagaagtttgcctagagccatagcagcccagctctagcaatgtatctcttcgcctgtaaagttctcgctgaccttagaaggtcacaccttagatgaggctcatggacttaggtaggctgggtagtaggccctgcctgctgatcctccctctctgatcagtggatggagtggcaccctcgggctcctgtcctgttggcgggcacagccgatggcaacacttggatgtggaaggtcccgaatggtgactgtaagacctttcagggccccaactgccctgcaacctgtggccgcgtcctccctgatggtgagagcagttatctcttcctgggggtacaggtggatccagtgacttggggttacttcagtcattgcagtctcctgccctatacttggtcttacctgtgactccagggggaatttggggaccccctacactatcctttctgctcattctttccttggcgtgtctgagtgtcctctgtcctctggttatccgtgccttggtccttagggaagagagctgtggtagggtatgaagatggtaccatcaggatctgggacctcaagcaaggaaaccccatccatgtactaaaaggtatcagagggggcggggtgttttccttggctttgagggaggagcctgagccaggtacaggactcaaaacctctgtccacctgcttgttctagggactgagggtcatcagggccctctgacctgtgtagccaccaaccaggatggcagtctgatcctgactggctctgtagactgccaggccaagctggtcagtgccaccactggcaaggtaagtagcctgggcttctttgtctggaccttctgcacctcccctgtcttttccctctttgtccaggtctgtgagcctcttttcttacctgtaggtggtgggtgtgttcagacctgagacagtcgcctcccagcccagcctgggagagggggaggagagtgagtctaactccgtggagtccttgggcttctgcagtgtgtgagtgtgagcagaaccttggcctggagacccaagagactgagggaggaggtggctttgaggaggcatctaggaattgagaccaaggaggagggaaggtatatgaatctaggacctcaagagccttctttgtgtcacttgcttcccagcatgcctctggccgctgttggctatctggacggaaccttggccatctatgacctgtctacgcagacactccggcaccaatgtcagcaccaggtgtgatgtctggggcttaattcctacttaggggctgttgtgctggctaaggggcagcctgtcctctgtggctcctcagggtgggttggggtcctctcttctcccttctgacttctgccctactatgttactgtgtcacactccgggacagtcgggcattgttcagctgctgtgggaggctggcactgctgtggtgtacacctgtagcctggatggtgttgtacgtctctgggatgcccggactggccgcctgcttactgactaccggggccacactgctgagatcctggactttgccctcagcaagtaagcattatgggctggcagctctagaaagtgggtctggtgtgaggtagcacaggtcctttgtgGAATGGGAGGCGATTCCTAAGGGAAACGGGACCCAGTGCCTGACTGTGTCCTGTAATAAAGGCATGCTGGGCCGTCCATCTGTGCAGTCACAGTCCTAGAACCCAGCCCTATCCCAGCGATTGGCAGTGAACTACCCAT
This mutation is a 2087 bp deletion beginning at Chromosome 1 position 74,280,718 bp and ending after 74,282,804 bp (GRCm38/mm10).
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
43353 | CTG GCA GCT CTA GAA AGT GG | Wild type Forward | A | |||
43354 | ACA GGA CAC AGT CAG GCA CT | Common | A | |||
43355 | TGG GGC CAG TAG TAT CTG TTC | Mutant Forward | A | |||
43356 | Fluorophore-1 | CTG GTG TGA GGT AGC ACA GGT | Quencher-1 | WT Probe | ||
43357 | Fluorophore-2 | CTA GTC CCT GGG AAT GGG A | Quencher-2 | MUT Probe |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
43353 | 0.40 uM |
43354 | 0.40 uM |
43355 | 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 |