Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.
>chr11:110072010-110072095 86bp AAGGAAGCAGCCTTTCCTCT GCTTCACTTAGCTGTGGTTAGACA
Mut= 80 bp
Wt= 86 bp
Fam=Mut
Hex=Wt
Wt Sequence (deletion in lower case):
TTAGTATTACTCCTTTCAGAGTTCTAAGAAATAGCTATATTTGTTGGCTAGTTAATGTTTAGACTCCAATGACGGCTAGCACCTCTGTTCATGGCATTTCTTCTTGTTGCCCTTCGATACTGCCTGGCTGACCTGAAGAGATTCTGGACTTTATGCAGatgttttttctaacatctgtaaagtgttagtttcctactttgccttgctatggtagctgtccacattgtatcattttctagcttaccctatcactacgtatggttcagctcaggaaaaatgctcacagtggcttgaacagtgtccaggtctcagtccacaccaaaggaagcagcctttcctctaggtaagatcaggctttaaaacactggtccaagtaccattgctgtctaaccacagctaagtgaagctcacatgccatcccttatatccttgcaagccaaatctgcgtaaagatttcagccagactaggagttagatagtaaagaaaaccattgctccttaaacagtttatctcttgctctctttctcagaatcagaaatcttacaaaagactacatacaaaagtctaagcgcacagaagctttgaaaggtaagtaactaaggtagaaaaatatggtttttagtcagttaacttttctctccatgagcaattctagactgtgtaagtggatgttgagtctcctgaccccccaaaccccacaactgagctaacattgacaggctgtgtgattttgagtattaactttgagtattaattttgatttaataagtgaatgaatgtatacttttataaattctagtcacactataaatacgtaggcactatatgagccctgaattaaacctacatttgtgattttataccttcatgctgttttcctcccagatctgacactggatgtatataaaggccagattactgcgatccttggtcacagtggagctgggaaatcaacactgttaaatgtcctgagtgggttgtgtgttcccaccaaaggtaagagacagagtctttcctctaagttggcaatcatctgtgcttttgttagacatttgagcattaaggtcagacagagataaactcccagctattgacacataatctctattactcttttataaaatgtatatgatattaactgaaatgtacagaattttagtattttagaatactcaaaattttatagaattaacagtttcaattgatattgatcacatttttctgtatagataaagtaaccttgaaaattagcaacttttaaaatatgACTGGAAAGGTCTCTCAATCTCACTGTTTTGACTGTAACTTATAACCTAGAATGGCTTTACCTACATGTGTGAGACTTTAGCTGGGCTATTCTCCCCTTCCCCTTCTCCCCTCCCTTCCCTCTCTATCTCCTTCACCCTCTCTATCTCCTTCATCCCCTCCCTCTCCTTCTCCCTCTTCCCTTTCTCC
This mutation is a 1110 bp deletion beginning at Chromosome 11 position 110,071,149 bp and ending after 110,072,258 bp (GRCm38/mm10).
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
46922 | AAG GAA GCA GCC TTT CCT CT | Wild type Forward | A | |||
46923 | GCT TCA CTT AGC TGT GGT TAG ACA | Wild type Reverse | A | |||
46924 | GAT ACT GCC TGG CTG ACC TG | Mutant Forward | A | |||
46925 | TAC AGT CAA AAC AGT GAG ATT GAG AG | Mutant Reverse | A | |||
46926 | Fluorophore-1 | TCA GGC TTT AAA ACA CTG GTC C | Quencher-1 | WT Probe | ||
46927 | Fluorophore-2 | CTG GAC TTT ATG CAG ACT GGA AA | Quencher-2 | MUT Probe |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
46922 | 0.40 uM |
46923 | 0.40 uM |
46924 | 0.40 uM |
46925 | 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 |