Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.
>chr8:79719381+79719489 109bp ACCACCCAAAACATTTGTCA GGCTGAGTTTGTTGTTTACTGA
Mut= 89 bp
Wt= 109 bp
Fam=Mut
Hex=Wt
Wt Sequence (deletion in lower case):
CCGGCATAGGATGGATCATTCTTGTGATTAGTTTATAAAAAGGATAAAAACTTGGACTTGTGCAAATAGAGACCTTATATAACTCCAAAGATGCTTTGAAAAAACTAACCACCCAAAACATTTGTCATAGTATAAACTTTAGTGTGTGTAAGGGattgagcccaggacctcccatttttaaaataaacagtctatcagtaaacaacaaactcagcccattaggggatttttagagttgaaatctggatgaaagaatagaattctgcttaaatttcatattctaactgtcttctatgtcaacatttctagacaaatatgtatggtaattttatgttttaaatatgtaactttttcatggaaatttgtcattttcactgtttctagggtttggagtggatcaattacgggatgacaatctagaaacttactggcaatctgatggctctcagcctcacttagtgaacatccaattcaggtaatccatttcttacatttttttatatagtgtaggttgtaagtggacaaggaaatctattttattttcataaaagttaagaaaacatggaaagtaaaatgaaaatacttaattgctttcagactctattaggtcttagagatttttttttttttttgccatccagATTGCAGGTAGCTTCAATGTATTCGGCCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCAGTAAGTCAACAGGTTCTCCAGTGCAGGAGTGAGGATTAAAAAGACAGATAACATTGCAGCGTGACCCCAGGCAATTCTGAGACTGAAGGCCAATTTAATATTTCCAAT
This mutation is a 487 bp deletion beginning at Chromosome 8 position 79,719,428 bp and ending after 79,719,914 bp (GRCm38/mm10).
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
43358 | ACC ACC CAA AAC ATT TGT CA | Common | A | |||
43359 | GGC TGA GTT TGT TGT TTA CTG A | Wild type Reverse | A | |||
43360 | GAG AGA GAG AGA GAG GCC GAA TAC | Mutant Reverse | A | |||
43361 | Fluorophore-1 | ATT GAG CCC AGG ACC TCC | Quencher-1 | WT Probe | ||
43362 | Fluorophore-2 | TGT AAG GGA TTG CAG GTA GCT T | Quencher-2 | MUT Probe |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
43358 | 0.40 uM |
43359 | 0.40 uM |
43360 | 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 |