Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.
>chr5:120499165-120499262 98bp GGAACAAAACTAGGGCTGGA CTGTGGGCAGACTCAGGTGT
Mut = 111 bp
Wt = 98 bp
Fam = Mut
Hex = Wt
Wt Sequence (deletions in lower case):
AGGTCGGGAGAGGAGGCTGGTTTAGTCTTTGTTCCTCTGCCCCAGCACTTCCTGTCTCAGGTATGTAGCCCAGCAGGCATGTAGGTACAGACATGCAGGCTAACATCTGTATGGGCAGCTGCTGCTCACCAGGTGCAGACAGGCAGGCCAACATCTGTATGGGTGGCGGCTGCTCACCAGGCTGTTTTCATGGTCTAAAACTAGAGACTAGGCAGGGATGGTGTCACACATCTGTGATCTCAGCACGTGGAAAGCTGAGGCAGGAGGATTGGAGTCCCAGGCCAGCCTGGGCTACAGAGTGAGACCATGTTTGAAAAAGGGACAGGAACAAAACTAGGGCTGGAGCAGttgcctgccacacgagggcctgaactccatgtgtagtgtaaggaataaaaagccacacctgagtctgcccacagccgtgggtctggctgtgtggctgtcttttcaggggtccctgctgctaactgactgtccttcttctcccctccaggtgggcctatctggacctgagcacaaatggcaggtacaatgacagcctgcaggcctatgcagctggtgtggtggaggcctctgtgtctgaggaggtaaggccagtgtgggagtccagggtcccagctgcctgcccctggccaagcccagacatcctgagggccgtcagtggggttgATGCATGTGTTGAGAAAGGGTCCCCATCTCCTGTCCTCCATTAGGGGAGAAAAAGAGCCCTGTTCTTCAAAGACAGCACATAGGCTGGACCTGGTGAAACACACCCACAATTGCATGACTTGGGAGGCTGAGACAGACTGCTGAGTTCAGGGGTAGCCTGGGTTAGAGACAAGAAACAACAAAAACTTCCAGGCTATGATTCATGCCTGTTGTCCTAGCACTGGGGGATGGAGGGAAGACAAGGGTCAGGAGATCAGAACCAGCTGTGACTACTCCCC
This mutation is a 324 bp deletion beginning at Chromosome 5 position 120,498,915 bp and ending after 120,499,238 bp (GRCm38/mm10).
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
49129 | GGA ACA AAA CTA GGG CTG GA | Common | A | |||
49130 | CTG TGG GCA GAC TCA GGT GT | Wild type Reverse | A | |||
49131 | CAG CCT ATG TGC TGT CTT TGA | Mutant Reverse | A | |||
49132 | Fluorophore-1 | CCA CAC GAG GGC CTG AAC T | Quencher-1 | WT Probe | ||
49133 | Fluorophore-2 | AGA TGC ATG TGT TGA GAA AGG GTC | Quencher-2 | MUT Probe |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
49129 | 0.40 uM |
49130 | 0.40 uM |
49131 | 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 |