Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.
>chr5:74482088-74482191 104bp AAGCAGCCAGCAGAGAGAAC TGCAGCTTTGCTTCTTTTGA
Mutant= 120 bp
Wild Type = 104 bp
Wt Sequence deletions in lower case t insertion in <t>:
CATACTTTCATGGAACAATGTAGCCTTTCTTCTCCCCTTTATCCTGAGTTTGTTGTCCCCTTAATTCCTTAATATCCTGCCCATTAAATGAATTAGACACCCTGTATCAAGTTAATACTTCC<t>cagtggcaatgatggtattagtgctcctatttcccagtaggaatttactctaagctgtatgtgtttccactgcagtgaggcctcagccaaggccctgtgggaagccttactgaactccaagcacaaagaggcagtgatggaagttcggagacatctagtggaagcagccagcagagagaacctgcccatcaagatgagtatgggtaagcccctgcctctgCGGAGGCAGCCCAGAAATCCTGTTTTCAAAAGAAGCAAAGCTGCAGCTTTGATATTGCCCTGGTGTCTGTCTATTCATTCCG
This mutation is a 220 bp deletion beginning at Chromosome 5 position 74,482,133 bp and ending after 74,482,352 bp (GRCm38/mm10).
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
46663 | AAG CAG CCA GCA GAG AGA AC | Wild type Forward | A | |||
46664 | TGC AGC TTT GCT TCT TTT GA | Common | A | |||
46665 | GTT TGT TGT CCC CTT AAT TCC | Mutant Forward | A | |||
46666 | Fluorophore-1 | CCC ATC AAG ATG AGT ATG GGT AA | Quencher-1 | WT Probe | ||
48173 | Fluorophore-2 | TAA TAC TTC CTC GGA GGC AGC | Quencher-2 | MUT Probe |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
46663 | 0.40 uM |
46664 | 0.40 uM |
46665 | 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 |