Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.
Mutant= 99 bp
Wild Type =116 bp
>chr17:36950289-36950404 116bp AAATAGCTATACCATGTTGCA GGCCTTTGATCTCACTTTCT
Wt Sequence (deletion in lower case):
TTGTTGTTGTTTTTGTTCCCGTGGATACAAGGGATCAAACTCAGGTCTTCATGCTTGCACCAAAAGAGCTTTACCAACAGAGATGTCTCCCCATCCCTGCTTTATTTTATTTTATTTTTTTCTGAGAAAATAGCTATACCATGTTGCAGAGGCCATTTCAgtctcccaagtgctgagattacatacctcagtctctttagaacatgaataagaggtgtgaagaagaaagtgagatcaaaggccagtcacaggaaagggagaaaataacattcatatattaatgccaccaagggtgatagcgttggttgtatcttagtttgccaatttgctggcaccttccctagtttaagaattgccttctttgcatactattggttatcaagaacagggattcatactctccacaaacaggctgtcctgcctctccaggggcatgatgtgtgttactgactgagccagggaagagaaaaaaggaggttccataggaaacaggaaaaatcaagaggaagtgagtcagatgggcccacacctcttttaacttttcaggagaatcagagcctaactatgaaacttcggaaacgcaagccagagaagaaggtggagtggtccagtgacactgtggacaatgagcatatggggcgacgctcgtcgaaatgtgagtgtgggtggcccagtgtccctggccaccatctcactgttgctttatagctctatcgagttcttggttggaaagaaaaataggtgggaataaaactatgcGGTACCAACTGCCTGGAGTTAGGGGTGGGGGCGGGGGCAAGCCATGGCAGAATGGGAGTCAGGTTTTTCCGACTCTCTCTTGAATTCCTGGGGGAAGAGGCTAAGGCAGCTGGATTCCAGAAAAGTAGGGAGAGGTTTTCACACAGATCTG
This mutation is a 599 bp deletion beginning at Chromosome 17 position 37,260,665 bp and ending after 37,261,263 bp (GRCm39/mm39).
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
68924 | AAA CCT GAC TCC CAT TCT G | Common | A | |||
68925 | AAA TAG CTA TAC CAT GTT GCA | Mutant Forward | A | |||
70005 | GGC CTT TGA TCT CAC TTT CT | Wild type Reverse | A | |||
70006 | Fluorophore-1 | TCT CCC AAG TGC TGA GAT TA | Quencher-1 | WT Probe | ||
70007 | Fluorophore-2 | AGG CCA TTT CAG GTA CCA ACT | Quencher-2 | MUT Probe |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
68924 | 0.40 uM |
68925 | 0.40 uM |
70005 | 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 |