Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.
>chr5:125431742-125431836 95bp TGTCATGAGTAAGCATGGAGGA TCAACTCCTTTCAACACATCC
Mut= 95 bp
Wt= 95 bp
Fam=Mut
Hex=Wt
Wt Sequence (deletion in lower case):
GCCAGGTTCCCTCCCACCTGAGCTACATCCCCAGCCCCACCCCCAGCCCCTCAAGAATGTGATAGCTTGGTAATGCTAACAGTTGCAGCACACAGACCCTTGTGCAGCACTCAGAGATTGGTAGGTGACCTTGGCCACCTGCAGGAGGTGGTGACCTCATTTGATATTTGTCATGAGTAAGCATGGAGGACGCCCTGTGACTTTTTGCCCCCTAGTgagactctgttcgtcttttcaggcaaggatgtgttgaaaggagttgatgaaagcaatgttctggtgctgcctgggaagagaaagaagaagaccaaggcgccgcccccatccaagaaagagaggaagcccctgacaaagaaggagaggaaggtgctacagaaggtcttagaacagaaggagaagaagagccaggtacacctcactgtggctggtctgcaccagctctccagatCAGGGGGCTGGAGGGGAGTTTGTCCCTCCTTGTTATCTCTGCAGCAAGATTGTCTACTAGAACTTCCTGAAAGGAGGGAAGTGTTTTGTGTCTATCCAGAGACACAGTAGCCACTAGCTACAGGTGGCATCATTGGGAGCCTCTGGGTGTGCTGGCCTGATTTCAAAATTAGGTACATAAAGAGATGTGTACGTAAAGCATAAAGGTATGCGTCAAA
This mutation is a 232 bp deletion beginning at Chromosome 5 position 125,431,557 bp and ending after 125,431,788 bp (GRCm38/mm10).
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
43990 | TGT CAT GAG TAA GCA TGG AGG A | Common | A | |||
43991 | TCA ACT CCT TTC AAC ACA TCC | Wild type Reverse | A | |||
43992 | TTG CTG CAG AGA TAA CAA GGA G | Mutant Reverse | A | |||
43993 | Fluorophore-1 | CTC TGT TCG TCT TTT CAG GCA | Quencher-1 | WT Probe | ||
43994 | Fluorophore-2 | TAG TCA GGG GGC TGG AGG | Quencher-2 | MUT Probe |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
43990 | 0.40 uM |
43991 | 0.40 uM |
43992 | 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 |