Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.
>chr2:13224647-13224768 122bp CTCTGTACCTCAGAAACAAG AGGGGCTGAAAGCAGAAATG
Mut= 121 bp
Wt= 122 bp
Fam=Mut
Hex=Wt
Wt Sequence (deletion in lower case):
GAGTTCAATGATAACAAGGACAGCTCCACAGGTACGGGATCATTTAAGAACTTGAGTGTACCCTCCTTCATTGGGGTTAGCTGTCTTCGGCTGGCAGTTCAAAGCAACCACAAGGAAGAAAATAACTTCAGCTGTTATGCTTGTAGTGTGGAAGTGTGTGCTGTGTTCTATTCCCTGAGAGGCTGCTGACAGCATGGGTCTCATGTCTTCCTCATTTCTGCTTTCCGCCCCTCTGTACCTCAGAAACAAGTCCTGCCAGGTAGTTTGATCTAATCAGACAAAAGGAAacacttcctgagaggctgctgacagcatgggtctcatgtcttcctcatttctgctttcagcccctctgtaactcagaaacaagtcctgccaggtagtttgatctaatcagacaagaggaaacacttccgttccacctggtcaagaatttttagccatgaaagtaagaaaacccacaaaatgcatgtggacagcactggggtttgaccttacttgcgttcaatagggaagtgtgtggctagcatcctttgccccttgaattcacatgctgcatctccatccctatagctgtgccaccaaatgtagcggaactgaagaacttggaggtactaaacttcttcaacaatcagatcgaggaactgcctacccagatcagcagcctccagaaactcaaacacctgaaccttgggtgagtattggaggagcttgaggaaatctccgccaggtaatgagctgaagccaatgcatgctgtgggcaagcaaGGGGAAGCGCTTATATGATTATCACATGGGAGTAGTGGGTTTCTAATTGCTTTTAATGTGTCAGGTTTAAGAAAAGGCTTATTGTTTTAATATTTCCTTTCAGTCTTCTCCATTAGTGTATATCCAGTTAAAGCTAATTGAGAAGCTAATTGTTGGCACAACAAGGAATCTTTTCATGATATTTTGT
This mutation is a 481 bp deletion beginning at Chromosome 2 position 13,224,231 bp and ending after 13,224,711 bp (GRCm38/mm10).
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
46951 | CTC TGT ACC TCA GAA ACA AG | Common | A | |||
46952 | AGG GGC TGA AAG CAG AAA TG | Wild type Reverse | A | |||
46953 | CTG ACA CAT TAA AAG CAA TTA GAA ACC | Mutant Reverse | A | |||
46955 | Fluorophore-1 | ACT TCC TGA GAG GCT GCT GA | Quencher-1 | WT Probe | ||
46956 | Fluorophore-2 | CAA AAG GAA GGG GAA GCG | Quencher-2 | MUT Probe |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
46951 | 0.40 uM |
46952 | 0.40 uM |
46953 | 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 |