Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.
>chr11:20240714-20240833 120bp CCTTAGACCAACTTCGTTTTCA GGAAAGGGCACTGTGTTGA
Mut= 119 bp
Wt= 120 bp
Fam=Mut
Hex=Wt
Wt Sequence (deletion in lower case):
GGCTTCATTACAGGAGATTTCCGAAATAGGATGAGTCTCGGTACTAAGCACTAGCAGCGGGCACCCTGAAATCCCCTGAACGCTCTGCTCGTCTTCCTTTTCTTAGTATACCAAGCAGTCGGTTTAACCGTAGGATCCTTAGACCAACTTCGTTTTCATTGATAATCTTCCCTATTCCCCCCCCCCCCCCagtcttgtgaagaaaactgttcttagaaacaagaagtctgcttggtttcaacacagtgccctttcccccttgttattgcagatggaggaagacaggcttcctgcctcccaggacctacttccagctcttcaagtctttggaactataactgtttgctcaggacaggaggctgacagtgaagacttccaggctacgcttgatccatctcaggtgttgggcctcagccagcaaccacacacctcaggtctccctctcccgccacagtggaagtccacggtgagcccaggtgctcctcagctttctagcagaagcatctctgcctcctcggtgggcagcagtcttcaggatcaccaagagaaggcaggacctcagagggcctcttttgccaatgtctcctctcccgagttgactgtaccccaggcagcccactctgtggtgggggcagggcctccgctccaggggtcagcacagcctctgacctctggcagtgatgctacaggcctgggtaagagacacctctcctttcaggcagaatactgggcctgtgcgctgccaaattctctgcctccttcccctaaccgccactccgcactctgggacccaaataaagagtatgaagatctgcttgactacacttacccactgaggcctgggcctcagctcccaaagcaacctgaaagtcatgtcctgactgagcctgttctgcaggactcaggtgtagatctggacagtttgtctgtctccccagcaagtactctaaagtcacccactaatgtctcccacaattgctcatcagcagaagtgcctactctgccattttctggagccagagagtcatgtcttaagcgctggcccttgggaatattccagaaacagggtggcacaagcttgtcatcctggaaccagcttgcatcaacccctagagccccaggcactgaagatgcttcttgggagaacagagaggcagccctgaggggcacagcggaggactgcctccctataggtgaggacctccgaatgggctctccccagctgaagacgaaggagaaagagccaccctttcccagacagaagagaggcaggcagcatgttagctgcccggcctgtgtgacgcctggatggccatcagaagaggaggtgggaagtgatgaggagtaccttgctctgcccaccaggctgactcaggtctctagtttggtgtcctactcaggtgccaggccctcctttgtgaacctacacactggggctgctgaggaacacagttcactacaagtctcagacagtgacaagccagcctcccccacattggactccagccacaggaagcatccttctggaacttcctttcaagggcctgtgggccagaacccctgcttcaggcattctatccagccccaggactctagaggcaaaagtagtctgatgagtaaccagaccctcggggtctcttcaaaaccactgaaaactcagcccgcctcaaaagccatgacggacaggcggctgttctccgagctagtagctggagagacacttcccaggacgacagatgagcaggagaaagcttctctagtgcagtgtgtgcaggtaaaaatgttcacggagaatcttctggaaggagtgatgtttccccagcccaccaacagcatgtctcggcaccctcaacatatatgaccccaaattagaggtatcgttctcctttagtgattgcaagaaaatggcagagaaatcaggtaacgcagcagcagtgttccgtgtgctttggtagaggctgatccacagttttactagtttactcactcatcctgcatctgcttttctgcagagaaagtttagtggtcggtaacggcccttgttccttagttccactctcaccttcagggctctgcattctgagcctagttctgatatcacatctggctgaggcacagccagttatggcattcattatcctggcagaggaaaacctggagtcttgggatagaggtggagacaaatgaccatctgaaagaaaactctagaaaatttgtatgccaaaatgctggtaattttagccttcggacaagttaaatagcagctcagtcagatgtgccatgtagacttctcttgttgtactgagccctgaacatgcctgaagtaggcagacttcatcactgagctcagtccagcccaccaattgttcttgtctccacagacattttgctgtcggctggaagagctgatctgctggttgtataatgtcacagatgttgctgacctcagtgctccacccaggaccagccttacaggtctcaagtcttctctgcagctttacagggtaatatgtagtcctggcttgtttatccacggggatgggagttaagtcacttttacaaaataaaatctcaagtaactggagtcttgtagctcagttgactattttatctgataaatcgaattaagatgtccttttcaaattctcagtgttgaacactataaaatgtactaagtgaggtaagaggataattacatacattggagccagaatgctttagaggagtttgcatgggattctggcagatccctcagggctagaatcctgggCTTTGATTTACAGACACTAATCAAGTATGACAATTAGTTAGGGTGAGCACCACAGCTATGCAGTGGCCACCACACCAGGCAGTGCAGACACAGAACGTGCCAGCCTCACCGCAAGTTCTTTTGGACTGTGTTGGTGTTTATGGGGTTGAGCTGAAACTACTGGGATCCAGGCACAGACAAGGTGATGTGAGACTTTGGGTTTTATTTCTGCC
This mutation is a 2580 bp deletion beginning at Chromosome 11 position 20,238,200 bp and ending after 20,240,779 bp (GRCm38/mm10).
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
46234 | CCT TAG ACC AAC TTC GTT TTC A | Common | A | |||
46235 | GGA AAG GGC ACT GTG TTG | Wild type Reverse | A | |||
46236 | CAC TGC ATA GCT GTG GTG CT | Mutant Reverse | A | |||
46237 | Fluorophore-1 | CTT AGA AAC AAG AAG TCT GCT TGG TT | Quencher-1 | WT Probe | ||
46238 | Fluorophore-2 | CCC CCC CTT TGA TTT ACA GA | Quencher-2 | MUT Probe |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
46234 | 0.40 uM |
46235 | 0.40 uM |
46236 | 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 |