Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.
>chr9:44317095-44317186 92bp TAGAAGTGGGGAAGGGCAGA CAGCAGAGAGCTGGCAATG
Mutant= 96 bp
Wild Type = 92 bp
TCTAGGGAGAGAAGAGTTTTTGTCTTTGTTTTGAGACAGGATTTTACTGTGTAGGTCTGCCTGTCCTGGAACTCACTCTGTAGACCAGGAAATCTGCCTGCCTCTGCCTCCAGAGTACTGGGATTAAAGGCGTGCGCCACCACCACCTGGCTGAGATTTTTGCTTTTGTCTAATTCATTGAGAAGCTTAGGGCCCCCAAAAGGACATCATCCCAAGGATGATAGAAGTGGGGAAGGGCAGAGCCAGGTGGGTGTCCTCcgcagtgcctgacactgttccctgtttcaggtgctgcattgccagctctctgctgaggaggtgcgcttccccatctctgtgacccagcagtcccccgctgccgtctccatggagacctaccacgtcactctgacactgccaccaacacaggtggagggaaatgggggaaactgagttgggcagagggcagttgagttggcccagcattttctccctgcttcctctctaccgttggattcgggaaggaagggtccgagtagttagcagggtggggggatggggtcctgaagaatggggtagatcacagtaatccttccgttcttgtctctagttggaagtcagcctggaggaaatccctgatgaggggctcctggtgtcctgggccttcactgaccgcccagaactcagcctaaaggtgcttcccaagttgcagactagggaggtaagaagggggcagcagaggagagactgaactggcgcaatggagcagggcggaggctccacctcttctcctctctgcagagagatgaggaacaaccagagctctcaacagttgaggaactgatcaaggacgctatagtcagcactcagcccgccatgatggtcaacctcagggcctgctctgccccaggaggcctggtgagtggacacccaaagggaaggtttgcgtgtatcccacctccaggttactatatcgggattatttctctttttactcccttttgctcttaggtacccagtgagaagccacccacgatgtcccaggcccagccatccatccccagacctacccgattattcttacggcagcttcgagcatctcacctgggaagtgagctaggaggtgagaattccgctgcccaggacatagcttctctattccgcggacttggtttcttccgtctgagcatcctctaagctctgcttcctgacaggtactgaggaactgtgctgtgccgctgagcttgacaaccccatgcaacaaaagtggaccaaacccatgagggcgggccccgaggtggaatggaccgaggacctagctctgtaagtcacactctgctcctcccctctgtccccatgggcagtcaggatgaagaaacttttccaggctagccctggcatgaggaagccagccttgttttcagtagcctgtagtgtggacctccactgtatgccaggccctggggacacagactaggagtagatacagccctacctctgggtggatcacagacccgaagcagagacacatagggaagaaaggacaaagttgccagttgagattggtgcacagcatgcCGTGGAGGCGCTTCAGTAGGGGAGATGACGAATTCTCAAAACGGACTAAAGAACTGATGAAATGTCATGGAAGAAGTCTATTTAAACCACGCCTTCAAGCAAGGGTAGCACCGCAACAGAATGTGAAAGTCTTCTT
A 1299 bp deletion beginning at Chromosome 9 position 44,315,851 bp and ending after 44,317,149 bp (GRCm38/mm10).
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
45127 | TAG AAG TGG GGA AGG GCA GA | Common | A | |||
45128 | CAG CAG AGA GCT GGC AAT G | Wild type Reverse | A | |||
45129 | CAT CAG TTC TTT AGT CCG TTT TGA G | Mutant Reverse | A | |||
45130 | Fluorophore-1 | AGT GCC TGA CAC TGT TCC CT | Quencher-1 | WT Probe | ||
45131 | Fluorophore-2 | TGG AGG CGC TTC AGT AGG | Quencher-2 | MUT Probe |
Component | Final Concentration |
---|---|
ddH2O | |
Kapa 2G HS buffer | 1.30 X |
MgCl2 | 2.60 mM |
dNTP KAPA | 0.26 mM |
45127 | 0.50 uM |
45128 | 0.50 uM |
45129 | 0.50 uM |
Glycerol | 6.50 % |
Dye | 1.00 X |
Kapa 2G HS taq polym | 0.03 U/ul |
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 |