Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.
>chr6:30614129+30614224 96bp TCTTCCAACCCTGAAACTTCC CATGTCCCTAACATGGCAGA
Mut = 116 bp
Wt = 96 bp
Fam = Mut
Hex = Wt
Wt Sequence (deletions in lower case):
CAGGTTCAGACCCCAGAGCTTGAGATGTGGGACTCTACATTGTCATCTTCCACCTACCCCATGGAAGGAAGCAAGCATGGCTCCTCTGCCCCAAGgttggggttattttatatcacctttcatggggggtgggtagcacaggaactgtcattggttccccagttccctttagtccatctgcccactccctcatcccttctgttatgtgccagggctccttcacttcagtgtcccctctcttccacactggctctgtcaatcgccctagcctccacagctttcctcacccttcccatcttttcccagagaccaggttcttcgagtcctggccaaaaatgagaagcagctgtcacttctcagggatctggagacccagaagccccagaaggtgaggattgttcctggggatactactgtcaccacatttgcacctcagttccttgtcttgaccccttacatcttccaaccctgaaacttccttcctgaggtctccaaaaaggatcactagattctggacaagtccaagGTGGCTTCTCTGCCATGTTAGGGACATGTCCCCATAAATTTGGGTGGCCTTTGTAAGCTGTGATTCCTTTAGCTTTCCCT
This mutation is a 431 bp deletion beginning at Chromosome 6 position 30,613,766 bp and ending after 30,614,196 bp (GRCm38/mm10).
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
50465 | CAT GTC CCT AAC ATG GCA GA | Common | A | |||
50466 | TCT TCC AAC CCT GAA ACT TCC | Wild type Forward | A | |||
50467 | AGA CCC CAG AGC TTG AGA TG | Mutant Forward | A | |||
50468 | Fluorophore-1 | AGG ATC ACT AGA TTC TGG ACA AGT CC | Quencher-1 | WT Probe | ||
51939 | Fluorophore-2 | ATC TTC CAC CTA CCC CAT GGA AG | Quencher-2 | MUT Probe |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
50465 | 0.40 uM |
50466 | 0.40 uM |
50467 | 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 |