Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.
>chr4:156250527-156250614 88bp TCCTCCTCCCACTCTGTTCA CCCTGATAAGCTCCCGTAGG
Mut= 86 bp
Wt= 88 bp
Fam=Mut
Hex=Wt
Wt Sequence (deletion in lower case):
CCTTTCTTCTCCAGTTTCGATGACCTACTTATTCACATTCCTTTTCAAGTTCACTTGCCTGGCTACAGCATGTAGGTGTTAAAGCAAAGTGCCTTCCCTGCTGATCCTCTAGCCTGTGACACAAAAGCAGGCTCAGGACATGTGAGAGTCTCCTCCTCCCACTCTGTTCATACCTTCACCAGGCCAATgccctgctcaccaagatggaaaggggaaatcctacgggagcttatcaggggggcccgctatcacctcccttctcagatctgcttagggtacgccaggaagtagcaactgcaacactgaggggtcccagtggcttggaagttcacctgccctcgtctacagcagaccatcgacggaagcaaggcctagttcagcgccgggaaggggcggttccagctgctgccacatctttctctgaaaggtattaagacgtgcttcagcaaggccccacatgctatgcttgttacttgagaaggccaattctttctttcccttgagtcttccatcacatatatgcataatgcattcaccacgttcaaggtccactgcaggaagctcctaaaactactgcaaagactcaatttggggggcaaaatatgcagaaatgcccactccctggcaagaatgccaaatgcaattttcttgtcaagcaactctgagtattccagcctagtagagctgccccgagagcagaggccctcCCCTCACAGGTGGTGGTGGTGGGATGTTGAGGAAGGCAAGGAGAAAGCAACCACAGAGGATTCAGAGTGAAGGACATACGCCCCCTCCATCCCTACCCTGCTATAAACTCCAGAGACATCATGCTCACAGTAGTCTCCGCGGCAGACGAGTTAAGCCAAGGAGAGCATTAGGCAG
This mutation is a 519 bp deletion beginning at Chromosome 4 position 156,250,058 bp and ending after 156,250,576 bp (GRCm38/mm10).
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
39037 | TCC TCC TCC CAC TCT GTT CA | Common | A | |||
39038 | CCC TGA TAA GCT CCC GTA GG | Wild type Reverse | A | |||
39039 | GCT TTC TCC TTG CCT TCC TC | Mutant Reverse | A | |||
39040 | Fluorophore-1 | CCC TGC TCA CCA AGA TGG | Quencher-1 | WT Probe | ||
39041 | Fluorophore-2 | CCA ATC CCT CAC AGG TGG | Quencher-2 | MUT Probe |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
39037 | 0.40 uM |
39038 | 0.40 uM |
39039 | 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 |