Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.
>chr2:26390853+26390950 98bp CCTAGGAAGCCCTGAGTGAG ACTGCCCACAGTGAAGCTCT
Mut= 95 bp
Wt= 98 bp
Fam=Mut
Hex=Wt
Wt Sequence (deletion in lower case):
ATGCTAAGCTAAACAGGCAGGCTGCTGCTGGCTATCTGAAGCTGGGCATGTGTCCATGTGAAACCAGTCTTCATTTAGACCAGCTGGTTATTTGTCCAGGCCTAGGAAGCCCTGAGTGAGAGACttcctctgtgaagagaggagaaccagtttcctctggttccaaagtatctcatggagagcttcactgtgggcagttgtggctcccagaaggccttcacacccaagaaagaggtagtggtagtgtctttgctggcatgaagggcctcagtgactgagttgttatctggtgtaaaccatgcacctgctattgtggcttatttcctttcagagacaccaccatgtatgctgtgtctgctgacagcaaaggcttggacactgtggtggatctgttggctgatgtggttctgcacccccgactgacaggtgtggctctgaggctgggtggtcaggaggagctttggaagggctataacttcctgtggggaggcactgttagagccgggcactcatgtgccaatcattggagagtgccagtgctgaaacaagcctgcagtgttctacacaatgtttcatcctttaatatatctaaacgagaccttaaagggtttactaaagttctgctcagaaatgtccctcatcccgtgtgTAAGCATGAATGCCATGCCTACAGAAGTTTCTCTTGCCTGGTCTCCTTTATCCTCCGAGTCTTTGAGTCCTGTTAGGATGGGAGAGGGCGCAGCA
This mutation is a 525 bp deletion beginning at Chromosome 2 position 26,390,877 bp and ending after 26,391,401 bp (GRCm38/mm10).
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
39535 | CCT AGG AAG CCC TGA GTG AG | Common | A | |||
39536 | ACT GCC CAC AGT GAA GCT CT | Wild type Reverse | A | |||
39537 | AGG ACT CAA AGA CTC GGA GGA | Mutant Reverse | A | |||
39538 | Fluorophore-1 | TCT GTG AAG AGA GGA GAA CCA GTT | Quencher-1 | WT Probe | ||
39539 | Fluorophore-2 | AGA GAC TAA GCA TGA ATG CCA TG | Quencher-2 | MUT Probe |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
39535 | 0.40 uM |
39536 | 0.40 uM |
39537 | 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 |