Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.
>chr2:32905609+32905732 124bp AAAGCCCTCGTCTGGAGTG ACAGAAACCTGGGGAATCAG
Mut= 118 bp
Wt= 124 bp
Fam=Mut
Hex=Wt
Wt Sequence (deletion in lower case):
GGGTGTGGTTTTCTGTGACGGCCACTCACCAGCTTCCTGTCAGGAATGGGTACCTACCTCATACAGGCAGCTCATGACCAAAGCCCTCGTCTGGAGTGCGAGAGAATAAGGTGACcagccccggtttgtgtgaaaggatatttgtgctttgtgtggacggtttttcaggcgggacttgcaggcctgattccccaggtttctgtggcaacctcacactctcatgcatccttcccttgcagagaaaaccgagaagatcctcacagagttccttcgcttctatgaggaccagtatggtgtctccctcttcaatagcatgcgccatgagatcgagggcaccgggccaccgcaggcacagctgctctggcgcaaggtgagaggcatagagagggtccagcctgggctctgtaccccttctgctatgggcctttgtcttctctgggccttagtttccctatgctgctggtttggtctctaagggccttcataggttgaggataaccaccccacacttgcttcttagtctccactaccagtgccacccctaccccactgccagtgttacctaccctcactgccagtgctacccccccactgccagtgtcacccctctcccactgccagtgccacccacccccactgccagtgtcacccctctcccactgccagtgccacctctaccccactgccagtgccacccctccctcactaccagtgtcacccctccctcactgccagtgccacCCACCCCCACTGCCAGTGCAACCTCTCCCCCAATCCCAGTGCAACCCCTCCCCCAATGCCAGTGCAACCTCTTCATTGTCACAGCTACATTCTGCAGTCTTGACAGCTCAGGCCACACACCTGTCAAGAGAGAT
This mutation is a 626 bp deletion beginning at Chromosome 2 position 32,905,645 bp and ending after 32,906,270 bp (GRCm38/mm10).
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
43508 | AAA GCC CTC GTC TGG AGT G | Common | A | |||
43509 | ACA GAA ACC TGG GGA ATC AG | Wild type Reverse | A | |||
43510 | GTG ACA ATG AAG AGG TTG CAC | Mutant Reverse | A | |||
43511 | Fluorophore-1 | CCC GGT TTG TGT GAA AGG | Quencher-1 | WT Probe | ||
43512 | Fluorophore-2 | AGG TGA CCC ACC CCC A | Quencher-2 | MUT Probe |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
43508 | 0.40 uM |
43509 | 0.40 uM |
43510 | 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 |