Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.
>chr9:21140676-21140794 119bp GATGCTGCAAGACGCCATC CTTGGACTCACAACCCTGCT
Mutant= 116 bp
Wild Type = 119 bp
Fam=Mut
Hex=Wt
Wt Sequence (deletions in lower case):
TAGTTAAACCTAGTCTTCAAAAAAAACATAGACATGTGGCAGCAGGGCACCAGCGGATGGGTAGCACGTGGCTGTGTGCTCTGTAGTCAGGGCCCAGGGTGCGTCCCTGACCCAGGCCTGTCTGGTCCCCTGTGGTAGCAGGgtaggagttctttctatgactggctgctgattgtccctgcctgcaggcccgggtggaacgcatggagcagtttcagaaggagaaagaggaactggaccggggctgccgggagtgcaagcgcaaggtagccgagtgccagcgcaagctgaaggagctggaagtggctgagagcgatggccaggtggagcttgagcggctgagagctgaggcacagcaactgcgcaaggaggagcggagctgggagcagaagctggaagacatgcgcaaaaaggagaagaacatgccctggaatgtggacacgctcagcaaagatggctttagcaaggtgggacactggcaagagtgctatggagtgcggtcaggctagctgggggtggttcggtggtggaggctagacatgcctgtaaagaccccaggtccacccctgtgtccccatcccagtaaactagaaaaagaattctgacaatgagagccaggcaaagccaccgggtggggtgcgaggacaggggagggacagacagacagacagtttgagcttgggattggctagtggaggccaacgaagcccatccataccctctgatctgcccgaaggcccgcggccatgcctccgccaccttgatgcacttagcgacgccaccgcctccgatgtggacaggatgggcgcggcggccacggcccccaaaccccaggcgcacccctgccttgccccagcccacttgccctctcaccctgtcacagagcatggtcaataccaagcctgagaaggcagaggaggactcagaggaggcaagggagcagaaacacaagaccttcgttgaaaaatatgagaaacagatcaagcatttcggtaagttcctgcacaaaggctaggttaggagtcaaggccagcaaaaggcttccaatggcctctgcgacatcttcctaacatcctctcctgcaggcatgctccaccgctgggatgacagccagaaatacctgtcggacaacgtccacctggtgtgtgaggaaacggccaactacctggttatctggtgcattgacctggaggtagaggaggtaagtgtgcccttcccagctgcggggactcccagacactgcgcctgctcacgccccgcccacctctttgtcctggcagaaatgtgcactgatggagcaggtagctcaccagaccatggtgatgcagtttattctggagctggccaagagtctgaaggtcgacccccgagcctgcttccggcagtttttcaccaagatcaaggtaatgagctcctgtgtaccaccaggaaatccacagtgcagtgcgcgatgtcagatatttattagaggggcttgaggtgccacggggatggccgtacatatgtatacttgtgtgtgtgtgtatacatacatacatacatacatacatacatatatatatgtatgtatgtatatgaaacgtgtgtggatgtggatgtggacccatatgattaagagataactttgtgtggttggttctccacctttctgtgggtccctgtggtaaaacaagctgttaggcttacatggcaaagcactttcacctactgaacacctagctgaccctatttgaggatttctttctattcatttactttaaatattaaataatttatatgtaatgtaaatatattgcataaacattatataagtaaatattaattttttttgttgctgtgtgtgtgcctgagtgagtttatgtgcatcattcgcatgcaagtgtcttcagaggctagaagagggcgttgggtccctggaggtggagttgctagggtttgcaagctgtcatgtgggtaatgggtaccaaacttggttcctctgaaagactggggaacctgagagcagggcagggtagaacaatccagaactgaggtttctgtaagaccttgctggaggacacctgaagatgtgataccaagctggtgctgttgagccgagccaggcggctggccattagcaggtggtctcacaagctagtaggatgggcgctgaatgcaggagacaaaggccaaatcttgaggtagccatggtggagttcaggaaaagcttcagggagccttcacctccctaaacatctggttatgaattgcagtgagcagtacccaccctcttccacagaccgctgaccaccagtacatggagggcttcaagtatgaactggaagcctttaaggagcgagtgcggggccgcgccaagctgcgaatagagaaggccatgaaggaatatgaagaggaggagcgcaagaagaggctaggccctggtggcctggaccccgtggaggtctacgaatccctgcctgaggtgtgactccccagggcccaggagggaggagggtgatgtatacccattgctgctccctgcccccgcccctctgctcccacccatcccaagcccaggccctcaccctggcccctctgtactcaggagctgcagaagtgctttgatgtgaaggatgtacagatgctgcaagacgccatcagcaaaatggatcccactgtgagtccctagtccctgtggcagatggcagatggccagggGGTGCAACCCTGTCATCCAAGAGCAGGGTTGTGAGTCCAAGGCCA
A 2,555 bp deletion beginning at Chromosome 9 position 21,140,717 bp and ending after 21,143,271 bp (GRCm38/mm10).
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
49594 | CTT GGA CTC ACA ACC CTG CT | Common | A | |||
52542 | GAT GCT GCA AGA CGC CAT C | Wild type Forward | A | |||
52543 | CGT GGC TGT GTG CTC TGT AG | Mutant Forward | A | |||
52544 | Fluorophore-1 | CCT AGT CCC TGT GGC AGA TGG | Quencher-1 | WT Probe | ||
52545 | Fluorophore-2 | CTG GTC CCC TGT GGT GCA G | Quencher-2 | MUT Probe |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
49594 | 0.40 uM |
52542 | 0.40 uM |
52543 | 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 |