Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.
>chr6:17834879+17834966 88bp AGCTGTACCAGAGAAGTATCACG GATAAAGCTAATGGGAACGAAAG
Mut= 83 bp
Wt= 88 bp
Fam=Mut
Hex=Wt
Wt Sequence (deletion in lower case):
TTTCAGCTAAGATGTTTTTGTTATTTGAGTATTATATTAACTTTGAGAAAAATAATTTGGAATGCACCTTAACAATATTCTTTTATCTATCACTATATTTATTGATTCTTTTAAAAACCACTCTTCCAGCAGAGAAAAACCATTTTAActaaataatattggtatttagttactaaaatctattacagttagaccaatttaaacatttttcaaaggcggatagaaaaagtaaacatatattaagggaatgtatcctgaaattacaagtagaaactgaggtcataaacacacattatctttctgttctagatatttgaatggtggtattttcgcaagtatggaacatctttcattgaacaagtctccgtaagccacttgcgcccccttctgggaggggttgacaacaactcttccaataattctaattccagtaatggagactcagattccaacagacaaagtgtctcaggtatggaacgtctcttacaaaattttcgtgaaattaacaaaactgttttaaaagattacgtttcagagaaaaactgtgtttaaatttcaagggaggtaagaaagctgtaccagagaagtatcacgcatgcatcccagggacagaaaCTATTCATGCTGTTAACTTTACTTTCGTTCCCATTAGCTTTATCATAACAAATGATAATAGCATTAGTTTTAAAGCAAATGTAGCATAGAGTATAGAGTATTACATAGAGTATCAAGTGCATTAGCTACCT
This mutation is a 467 bp deletion beginning at Chromosome 6 position 17,834,456 bp and ending after 17,834,922 bp (GRCm38/mm10).
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
40946 | AGC TGT ACC AGA GAA GTA TCA CG | Wild type Forward | A | |||
40947 | GAT AAA GCT AAT GGG AAC GAA AG | Common | A | |||
40948 | TTT AAA AAC CAC TCT TCC AGC AG | Mutant Forward | A | |||
40949 | Fluorophore-1 | ATG CAT CCC AGG GAC AGA | Quencher-1 | WT Probe | ||
40950 | Fluorophore-2 | AAC CAT TTT AAC TAT TCA TGC TGT TAA CT | Quencher-2 | MUT Probe |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
40946 | 0.40 uM |
40947 | 0.40 uM |
40948 | 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 |