Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.
>chr11:76840606-76840718 113bp TGAAAGATCCTGTGGAGGTT CCACCATGAAGATTTCCAGA
Mut = 114 bp
Wt = 113 bp
Fam = Mut
Hex = Wt
Wt Sequence (deletions in lower case):
GAACTGGAGGATAGAGTCAGTGCCTAGCATGTGCCCAGGCCCTGAGTTCATCCTCAGGACTGATTAAGTGAATGATTGATTGATTGAATGAATGAATGAATGAATGAAAAAAGAATAAAGGAAGAAAAAACTGAGTTTATTTATGAAAGATCCTGTGGAGGTTTTAAATGCTTGGTATTGTCTGTGCCTTctaagggtgatatgtaatttgattatgttttcaaaggtttgtgctttctggaaatcttcatggtggctcagtggtagcaagctatccttttgatgattctcccgagcataagaccactggactctatagcaaaacctccgatgatgaagtctttagatacttggcaaaagcttatgcctctaaccaccctataatgaaaactggcgaaccccattgtccaggagatgaagacgagactttcaaagatgggatcacgaatggcgcgcactggtatgacgtggaaggtatgcaaagcactgactgttgcatttccttttgttggtttagctttcttacatgtattctgaatgcaggaggaaatgtgcaatttcacttcttaaacaggttatatctttgtatgtctgaagtttccaaaactacagcaaaatttaaagacgaccacagaaatatgtactatctccagttgacctcccctgctgatctCTTAACCCATATGCTCCACCTCCCCGCCTACACATGCCCCCCCCAATTCTTCCCCAGACCACTCAAAAATATGTAGCTGACAGATCCCTTAAGTCCCTCAGCACTTGTTCTTCCAAGCACCATTCCCCCACCTGAGAG
This mutation is a 483 bp deletion beginning at Chromosome 11 position 76,840,189 bp and ending after 76,840,671 bp (GRCm38/mm10).
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
50890 | TGA AAG ATC CTG TGG AGG TT | Common | A | |||
50891 | CCA CCA TGA AGA TTT CCA GA | Wild type Reverse | A | |||
50892 | TTT GAG TGG TCT GGG GAA GA | Mutant Reverse | A | |||
50893 | Fluorophore-1 | AGG GTG ATA TGT AAT TTG ATT ATG TTT TCA | Quencher-1 | WT Probe | ||
50894 | Fluorophore-2 | CTT CTT AAC CCA TAT GCT CCA CCT | Quencher-2 | MUT Probe |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
50890 | 0.40 uM |
50891 | 0.40 uM |
50892 | 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 |