Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.
>chr4:24792818-24792933 116bp AGGGCAAAATTGAGAACAGG GCCCTGTTTATGCCTTTCA
Mut= 111 bp
Wt= 116 bp
Fam=Mut
Hex=Wt
Wt Sequence (deletions in lower case):
AGCAAAATCAAAACCAAAATAAAAAACCCCAGGGAGCTACCAAGAGAAAAAGTCCCAGGATAGGGCAGAGGAACAAATTAGAGTAAAGGAAAACAAAGAAACAAAAAACAGAGAGGGCAAAATTGAGAACAGGAAGGTCAGACTCACAGGGCACTAGAAGTAGCCTCCCCAAAGCccaacgggtgagagagccccaggaagctgagccggtgaaaggcataaacagggcttcattactctcctttgtagtattcaagaaatgctgacgggccagaggctgtgccactcggaaactcacaatgacacggtcctggcggcgctgaatcagcaaagaagtgatggcattctctgcgacgtgaccctgatcgccgaggaacagaaattccacgctcacaaggcggtcctagcagcctgcagtgactacttccgggtaagtgaaggtggatgtgtttgtcttaggatgtcagtgcacagaggacagcctgatctttcctaggccgtgagccaactacatggcGGAAGGCAGATTTGTAAGTGTTGAGAACCTGTGTACCTTCctggacagaacTGAGTCCAGGACCCTGGTGGGACATTCACTGGACTGGGCTGGGTGGCTGCAATGAGAAGCAGACCGTTGCATCTGACTAATACATTTAACAAGCACTGAATGGTGTGGCAGTGACATGCATGGCATATGAAGATATGGTTCTTTTTTCCTAGAACTTGAAATTTGATTTCCAGCTGTGAGTG
This mutation is a 342 bp deletion beginning at Chromosome 4 position 24,792,530 bp and ending after 24,792,871 bp (GRCm38/mm10). There is an additional 11 bp [CTGGACAGAAC] del 40 bp after the del site.
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
44093 | AGG GCA AAA TTG AGA ACA GG | Common | A | |||
44094 | GCC CTG TTT ATG CCT TTC A | Wild type Reverse | A | |||
44095 | CTG GAC TCA GAA GGT ACA CAG G | Mutant Reverse | A | |||
44096 | Fluorophore-1 | CGG GTG AGA GAG CCC C | Quencher-1 | WT Probe | ||
44097 | Fluorophore-2 | CAA AGC GGA AGG CAG ATT T | Quencher-2 | MUT Probe |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
44093 | 0.40 uM |
44094 | 0.40 uM |
44095 | 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 |