Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.
>chr17:46260812-46260929 118bp CTGCTGCACTCTCCTCTCAG GTCCCCACAATCCCTGACAT
Mut= 104 bp
Wt= 118 bp
Fam=Mut
Hex=Wt
Wt Sequence (deletions in lower case):
TCAGCTACCAGACGCACTGAGGCCTTGGAGCGGGAGCTGGAAATAGGACAAGACTGCCTAGAACTGGAGTTAGGACAGAGCCGTGAGGAGCTGGACAAGTTTAAGGACAAATTCCGCAGGTGCGGAACAAGGGGCCAATCGTCTCCCATCCGTGGGAGCACCTTCCTGTCCATACGTTAGCTGAAGAGCCTCAGGTCCccctgactttatttagtaacccagatcttggaaggagagggaagttcctagactcccatgtggaggagggttggtggggcctgaggaacaggccttaaccagggctctgcctttctctgccttgttggcatgtgtaggctacagaacagctacacggcatcccagcggaccaaccaggaactggaggacaagctgcacgcactggtactgtgctgggagggtggtgtggccacaacatagcgcgggctaggacttccgggggggatgccactcaccctggggcctggggaggagagggaaggcagggcagtggtggggagggcgcggtgtgctgcgccaggcgggacagccctaatgctcacactgtctctcttctctctccgtctccctccctcatgctgccaccgctgctgctgcactctcctctcaggcctctcttagccacagctggatttttgcagttgcgtcTGAGTTTGTGTGTTTTCCCTGGTCCTGGCCTCCTTGGTCCATGTCAGGGATTGTGGGGACCCTGGCCATTTCCCTCTGCCCCTTGACCACACCCTCTCTTTTCCTGTCATGTGAGACCCAGAGTGCCTCCCTCCTGGGAAAGGGCTAGGGCTGAGGTTCTGCTCATGGCTCTGGCAGAGGCACTGAGTGATAGCCCTCAGCAGTCCCAGCCTTGGCTG
This mutation is a 468 bp deletion beginning at Chromosome 17 position 46,260,872 bp and ending after 46,261,339 bp (GRCm38/mm10).
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
64364 | CTG CTG CAC TCT CCT CTC AG | Wild type Forward | A | |||
64365 | GTC CCC ACA ATC CCT GAC AT | Common | A | |||
64366 | GGA GCA CCT TCC TGT CCA TA | Mutant Forward | A | |||
64367 | Fluorophore-1 | CTC TCT TAG CCA CAG CTG GAT T | Quencher-1 | WT Probe | ||
64368 | Fluorophore-2 | AGC CTC AGG TCC TGA GTT TGT GT | Quencher-2 | MUT Probe |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
64364 | 0.40 uM |
64365 | 0.40 uM |
64366 | 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 |