Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.
>chr11:77408144+77408235 92bp ACTTACCAAAACCGAACACG GAAAAATCCATTCCCAGGAC
Mut = 90 bp
Wt = 92 bp
Fam = Mut
Hex = Wt
Wt Sequence (deletions in lower case):
ATTTAGGTCTCCCAACTTTTGACTCTCTTGCCTTGGTGGCATGCTATGAGATGctggcttaaaggggggaaaaaagggttaaatgatttaaaatttctctaatttttttgtttctttgttatctgttaacctcaggctgtaagactggaaagtacttaccaaaaccgaacacgctatatggtcgtggtttcaactaatggtagacaagacactgaagaaagcattgtcctgggaatggatttttcttctaatgacaggtatgataccataaaaaatggggaaggggcatgaaaactactttttaagagaactacttttaaaataacctgttacagttattaaagctattgcaaaagtgtcttgtataattaagcttttaaaattcaagataaagTAGGGCTAGAAGGTAAGAGGATGAGGTTTGTGCGTTTGTGTTTTATCTTTTTGAGACAAGATCTCTCTGATGTAGACCAGGCTGACCTTGAACTCAGAAATCAGCCC
This mutation is a 341 bp deletion beginning at Chromosome 11 position 77,408,044 bp and ending after 77,408,384 bp (GRCm38/mm10).
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
52082 | ACT TAC CAA AAC CGA ACA CG | Wild type Forward | A | |||
52083 | GAA AAA TCC ATT CCC AGG AC | Wild type Reverse | A | |||
52084 | CCC AAC TTT TGA CTC TCT TGC | Mutant Forward | A | |||
52085 | GAT AAA ACA CAA ACG CAC AAA CCT C | Mutant Reverse | A | |||
52086 | Fluorophore-1 | ATG GTC GTG GTT TCA ACT AAT GG | Quencher-1 | WT Probe | ||
52087 | Fluorophore-2 | TGG TGG CAT GCT ATG AGA TGT AG | Quencher-2 | MUT Probe |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
52082 | 0.40 uM |
52083 | 0.40 uM |
52084 | 0.40 uM |
52085 | 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 |