Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.
>chr4:59689918+59690010 93bp GCAAGAAAAGAAGCTAACAGCA CACTGCATAGGAACAGGGATG
Mut= 111 bp
Wt= 93 bp
Fam=Mut
Hex=Wt
Wt Sequence (deletion in lower case):
TGAGCAAGTGAGCCTCCTCTCATGATGGGAAGAGAGTACACCATGAGGAAACGTGCCCCCACTCACTGCCCTGCTTTTCTCTCTTCATTGTTTCTTTATTTGCAGTTGTTGTTGTAGCAAGAAAAGAAGCTAACAGCATTCTCTTGAAACTCTTATcctgtgtggacagtcggctgtcttaccttttccatccctgttcctatgcagtgataatatctacatgcagcttacaagcacatttccttttttattccaaatgattatagttggaaaaaaaaatggactgtgatgtgggaatgtcttgggtaataacagtttcttgttttgattatttccttttaggagagacactgctgatcctgtaacatggaggattgtcttcatacctcatctgagaatctgtccaaattggtcagctgggcccacagccatgggaccatttgcagcctcattccaaacctgaaacacttgctttctgaaggttcccatgggaacctgactgcaatgtggggctgtagtgccggccatgcgtaccactggccactaacagctacttgcagagctgggtcccaagagagggtttgtttccaggacaaccgaagttttaattctgatagtcccagtataattggggtgccttctgagacacagactagccctgtggaaaggtaccctgggagacctgtgaaggcaaagctggactgtaatcggaccagagactcttgtgacttctcttactgcagtgagccctctgaactggatgaagctgtcgaggagtatgaagatgaaaacacactgtttgacatggtttgcgagtcttctgttacagatgaggacagtgactttgaaccccaaacccagagacctcaaagcattgctcgcaaaaggccaggaatcgtcccatcttccatccattcaagttcccagggacagatggtcgatgaatgcagtaatgatgtcatcatcaagaaaatcaagcaagagattccagaagattattacattgtggcaaatgcagagctgactggaggagtggatggaccagccctttcattgacacagatggcaaaacccaaaccccagactcatgctggtccctcctgtgtagggtctgctaaactgattccccatgtaacatctgccatcaacacggaattagacccacacattctgtcagcctctccctctgttatctccagaccgatcatcccaaagactgctagggtatctctggcttcaccaaacagaggaccccctggtgcccatggcactgcccaccaggtgaccatgcagatgcctgtgagcacatcacatcctaacaaacagatcagtatccctttgtctgctctgcagttgcctggacaggatgagcaagttgcttcggaggagttcctgccccacttgcctagccaggtctcatcatgtgaggtagccctttctccctctgttaacacagagcctgaagtgagctccagtcaacagcagccccctgctgctccaaccataaccactgaagccaccgcgcagtgcattccaggtatggaactcgaggtgactgtaagtccctcatccgcgcgtacctaagaatTCTGCGCCGTCAGCTCTTGTAAGACAGTTGGTTTCGTTTGCTAGGATGCAGTCATTTTCATTCTCAGGAAGGTAGAAATAATGAAATAAATTACATGTAAGGAATACTATGAAGGGATGCCATCTGAAGGAGGCTGTCCATTCACCAGATGCACCTTAGAGATGGCAAATA
This mutation is a 1432 bp deletion beginning at Chromosome 4 position 59,689,958 bp and ending after 59,691,389 bp (GRCm38/mm10).
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
45715 | GCA AGA AAA GAA GCT AAC AGC A | Common | A | |||
45717 | CAC TGC ATA GGA ACA GGG ATG | Wild type Reverse | A | |||
45718 | CTT CCT GAG AAT GAA AAT GAC TGC | Mutant Reverse | A | |||
45719 | Fluorophore-1 | TGT GGA CAG TCG GCT GTC T | Quencher-1 | WT Probe | ||
45720 | Fluorophore-2 | TTA TTC TGC GCC GTC AGC | Quencher-2 | MUT Probe |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
45715 | 0.40 uM |
45717 | 0.40 uM |
45718 | 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 |