Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.
>chr13:105250481-105250588 108bp ATTTCCCTTTACTACCTGTAGGC GGAACATTTTGGAGTGCTGA
Mut = 105 bp
Wt = 108 bp
Fam = Mut
Hex = Wt
Wt Sequence (deletions in lower case):
CATTTGCCGTCCTGTCTGCTAATCAGAACACAGAAGGCTAGCCTTTTCAGTAGCAGAAATGAAATGTGCATGTGATCGGCTTTTTTGTTTTTTTTTTTTCTAAACCCATTCTCTGGTCACTTTACAATGACAGCATTGTCTTTATTTCCCTTTACTACCTGTAGGCCCagtggacagttgggaaactgaattgtcctttctgtggggcccgattagggggctttaattttgtcagcactccaaaatgttcctgtggccagcttgcagctgtacatctctgcaagagccggactgatcaccaagcagcacagggagggagactaatgagaccagcgctgaaacatttgccacatcccggagtcccgtcaggttgtgataaggaaactctgctgacaggtggtggctccaaaaccagaaaccactggcttttaagcatggcccgaaacagtaacggcctcggaagacttacagaagcactctgcctggaggtgcgagcgacgtattttgagatgaagaatgaaaaactgctcttcaaagcctcggacccaaaatgccagccttttgttcctcagcctgacaccggcaggtgtccttcaagagcgtctcacagaaagtcacacagtttggacctgaacatcagtgagaagctgattttattacccactttatatgaaatacatcgtaagcctactgcctatcccaggctaaatgaaacggggcccattgacctttcaggcttggctttaccgtgtagtaacagtagctgctcctttcaaagcccacctagttttgatccgaatatgctgctgcacagactttcagtggctccccatgagacccaggcacaaaggggaagagaatgccagtgcggcctagaagcttcttcagtctactcggatcacgctaacgctaacagcctgccattcctgatggatctgccctcagcagggaggagcgtgctggaggcctcggaccaagaggagcacctctcccagctggacttcctgcgctctgccagcttccccttgggcaccattaaccacaggctgaacaacagggagagaagcaagttgaggactctgcgaaggcagcagaggcgcgaacgatggctacagaagcagGTAAGCGCTTTGCAACCAGTTTAGCAAGAAATAGTGTCCCTAAGTACTGGGGTCTGTGGCACAGAGGTCAATGTGAATGTACGCATTCTGAAATCTTAGTCTTAAGTTATGAGATGATGTGTCAGTCTGTGT
This mutation is a 953 bp deletion beginning at Chromosome 13 position 105,249,611 bp and ending after 105,250,563 bp (GRCm38/mm10).
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
49607 | ATT TCC CTT TAC TAC CTG TAG GC | Common | A | |||
49608 | GGA ACA TTT TGG AGT GCT GA | Wild type Reverse | A | |||
49609 | CAT TCA CAT TGA CCT CTG TGC | Mutant Reverse | A | |||
49610 | Fluorophore-1 | CTG TGG GGC CCG ATT AGG | Quencher-1 | WT Probe | ||
49611 | Fluorophore-2 | CGC TTT GCA ACC AGT TTA GCA | Quencher-2 | MUT Probe |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
49607 | 0.40 uM |
49608 | 0.40 uM |
49609 | 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 |