Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.
Mutant= 98 bp
Wild Type = 110 bp
Wt Sequence (deletions in lower case):
TGGATGGATGGATGGATGGGTGGATGGATGGGTGGACAGATGGATGGATAGATGGATGACTGACTTCCTATTTAGCTGAATTAATGGCTGACATCTAAGTCTCTGGGCCTAGAAGATGTTGGTGTATCCTATCTGTTCGGGCCATCTTGGTCAGTCCTGCCTATGTCTGTATGAACTCATGTCAGCTGTTGTCCACgagtggcaatcaagggcttcttctctcccttgctcccatccattatattcttcagtccctcacctcctcctcccttccctcccaggctgtgccttcgtgaagttttcttcccacacagaggcccaggctgccatccatgccttgcatggcagccagactatgccggtgagtgagagctgcgcttggctgaggcggggctgggtgcaaggtgggtgggagagatgggtggttttggcatcttcccttcatagactgcttcagacaggaggctggatgtgcagcccaggctgtgatcagagctgtgtgtgaccagacactgtcctggggcaagtgtagttcttagcttgtggcagtaacaCAGGCTGAGAAGCAGGGGCAGCAGTTGGATGATGTGTAGAGATCATGGACCCTGTTCTAGGGCAGCTGGAAGCCACGGTGGGTGCACAGAGTGAGGGGAGCTGCCAGGTCATTTGCATAGGTCCTTCAGGTCAGGTCCGCCAGTGAACAAGGGACAGAAGGACACTAAGAACCTAGAGGGGCAGGT
This is a 358 bp deletion beginning at Chromosome 10 position 81,305,085 bp and ending after 81,305,442 bp (GRCm39/mm39).
chr10:81469210-81469319 110bp GTGATCAGAGCTGTGTGTGACC CTCTACACATCATCCAACTGCTG
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
59177 | GGC CAT CTT GGT CAG TCC T | Mutant Forward | A | |||
59178 | Fluorophore-1 | AGC TGT TGT CCA CCA GGC TGA G | Quencher-1 | MUT Probe | ||
59179 | CTC TAC ACA TCA TCC AAC TGC TG | Common | A | |||
59180 | GTG ATC AGA GCT GTG TGT GAC C | Wild type Forward | A | |||
59181 | Fluorophore-2 | ACA CTG TCC TGG GGC AAG TGT AG | Quencher-2 | WT Probe |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
59177 | 0.40 uM |
59179 | 0.40 uM |
59180 | 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 |