Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.
>chr17:45426056-45426199 144bp GCTTGTCATTGGGTTTCACC GCTCTGGCACTCTCTCCTTC
Mut= 144 bp
Wt= 144 bp
Fam=Mut
Hex=Wt
Wt Sequence (deletion in lower case):
CACACTCATCTTAGGGGGCAGAGAGAGAGAGAGAGTGTGTGAGTGTGTGTGTTTGTGCACGAGGCCCCATCGCCTGGGCTGCTGAGAGTTTATTAGGACCTTCTTGTTCTGTTCCAGCAGTGTGTTTGTCTGGATGTTCTGCATTTGTGAGGAAGGGCTGCTTGTCATTGGGTTTCACCATGCGTGGAAATACCGAAATTAGAGAATTTTAATGCCGttttattcacgggaagcaattgtaaagatgctgttttccatagtgctcatctgttgtttattttgggaaggagagagtgccagagctagaacttcggtaattctcatactaaatggtgtgatacgggactaactcatctgtgattttttttttcccttcaagggacaaaactgcccaaagggacaatgaagaagaaacaacagatgacccccgaaagcttaagcctggagagatagatccaaacccagagaccaagccagcccggcctgacccgatagacatggatgagggtgagtggatgctggggagtgctcagctcactgtacaggactcaggccttcttggttctcctgtcctgactgttagagacccagttctgtcagtggttattcgaggaggcgtctccccgagtgtgtctgtcagtggttattccatctccccgagtgtgtctggtcctgtttttattagactgTTTTTTTGAGGATGCCCCTTATTTTCTGCTTCTTCATTTCATTTAATGAATCGAGCAGTGAAGTAGGCAGATGTCCTAGAGGCGTGAGCCTCGAAGGATCCTGTCTCATTCCCTTTGTAGAAGGAGAGGCGACAAGCTAGCATGTCGGGCCTTGCTGTTTCTTTGACTTTAGGGTGATTTCCTTTCCAGGAAAGAGCTGTGCTACAAGAGCTGTG
This mutation is a 344 bp deletion beginning at Chromosome 17 position 45,425,670 bp and ending after 45,426,141 bp (GRCm38/mm10).
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
47547 | GCT TGT CAT TGG GTT TCA CC | Common | A | |||
47548 | GCT CTG GCA CTC TCT CCT TC | Wild type Reverse | A | |||
47549 | CAC GCC TCT AGG ACA TCT GC | Mutant Reverse | A | |||
47550 | Fluorophore-1 | CAC GGG AAG CAA TTG TAA AGA T | Quencher-1 | WT Probe | ||
47551 | Fluorophore-2 | CCG TTT TTT TGA GGA TGC C | Quencher-2 | MUT Probe |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
47547 | 0.40 uM |
47548 | 0.40 uM |
47549 | 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 |