Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.
>chr5:110775322-110775426 105bp CAGGACTGGCTAGCTGAGAGA TCCAAACGCATGCTCATAAC
Mut= 100 bp
Wt= 105 bp
Fam=Mut
Hex=Wt
Wt Sequence (deletion in lower case):
CATCCCAGCAGGGAAGGAGGGATTGTGGCAAGACGGTTGCAAGTTGGAGGTTACTTTGGTGTACATGAGTTCGTTCCAGGCCAGCTAGGGTCACATATGGAGACCCATTCCCAAAAGCACCCCCCTCTTCCACTTTAGGACCAGGgtgtgagggttgtgttggaggatgaggcccctaacttacccgtcctctctgctctgttttctttgcagggtgtgtctgcagcagggcaggtggtatccctaaaggtgtggctgattgatgacattctggacaagatcaacagccaccttccatcccacattcggattctgggtaagcttccctgtgtgaccacctgagtgctctcatatggacagaggctgtgcttgctctggccaagcaggttctcagcggggtgctgtgaaggttcagtgttgctcctcaggactggctagctgagagagaacttccatggcctttggctgtactgttctaatagtatGTGGGTACTTTTCTCATCAGCAGTGGTTATGAGCATGCGTTTGGAATCGGGCTGGGTGCCTGTTGGGGCAGGTGGCACCAGAACCACAGTGTCAGGATGTCCCACCTTCGAGGTGTTTCTGCTCACCTGGGTTTCCTACATCCTGTCCCCCAGGATTGAAG
This mutation is a 340 bp deletion beginning at Chromosome 5 position 110,775,367 bp and ending after 110,775,706 bp (GRCm38/mm10).
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
40556 | CAG GAC TGG CTA GCT GAG AGA | Wild type Forward | A | |||
40557 | TCC AAA CGC ATG CTC ATA AC | Common | A | |||
40558 | TCA CAT ATG GAG ACC CAT TCC | Mutant Forward | A | |||
40559 | Fluorophore-1 | ATG GCC TTT GGC TGT ACT GT | Quencher-1 | WT Probe | ||
40560 | Fluorophore-2 | ACC AGG GTG GGT ACT TTT CTC | Quencher-2 | MUT Probe |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
40556 | 0.40 uM |
40557 | 0.40 uM |
40558 | 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 |