Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.
>chr4:12077432-12077550 119bp TGCCTTTCAAGCAAATGATG TCCACTGTGCAAGATATAAAGGAGT
Mut = 138 bp
Wt = 119 bp
Fam = Mut
Hex = Wt
Wt Sequence (deletions in lower case):
ACTTCAGTGAATGGTGTTATGTACTGGTACATGTACGTTTATAGGTTTGGTGTCTAAAAATAAAATTTAGTGTTGTGTGGGGAATATGGATTTGGAGGCTATTAGAAGCCCTCACAATTTTGACAACCTGCCAGCAATCTGTGAAGACACTCTCCTCATCCCAGCATGTAAGGAAAGATGTTGAGAAATTTGGCttagggaaaatgttggtttgctgttatagtcacttagaaaactagttctatgtgttttcagttggaatggagaagattttttttggtatattttattatatatatgcttggcatgcctttcaagcaaatgatgctactttgagaattattattacactgataattacagggacatgtgaatatttcagagtatataatacaacactcactcctttatatcttgcacagtggaaagaaatgttagtgggtctttgttagcccaagcaacttgcgagctttgtgatgaaagtgaaaactcatttacaaaagccaatgctttaggaaccaggtgagtagtgtactgaaaacaaaattcattgatgtcctacttttatgtgccatctgtgtatcccaagaaaaaatacaggtagtcattaaaggggcTGAGATTCAGAATCCATGAAAACGTGGGCTCAAGTCTTAAATTAGCTTTCTAATAATTTGGTGTTAGATAGTTAGAATCTCCTGTATTTAGAACGGAAGTACACTCTCCATTGCATAGGTTTCATAAGCTTCT
This mutation is a 423 bp deletion beginning at Chromosome 4 position 12,077,241 bp and ending after 12,077,663 bp (GRCm38/mm10).
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
48910 | TGC CTT TCA AGC AAA TGA TG | Wild type Forward | A | |||
48911 | TCC ACT GTG CAA GAT ATA AAG GAG T | Wild type Reverse | A | |||
48912 | AGC ATG TAA GGA AAG ATG TTG AG | Mutant Forward | A | |||
48913 | GAG TGT ACT TCC GTT CTA AAT ACA GG | Mutant Reverse | A | |||
48914 | Fluorophore-1 | CAC TGA TAA TTA CAG GGA CAT GTG A | Quencher-1 | WT Probe | ||
48915 | Fluorophore-2 | TTG GCT GAG ATT CAG AAT CCA | Quencher-2 | MUT Probe |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
48910 | 0.40 uM |
48911 | 0.40 uM |
48912 | 0.40 uM |
48913 | 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 |