Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.
Mutant= 107 bp
Wild Type = 82 bp
>chr2:101643577-101643658 82bp AAAGAGACTTTGGTGCATATCAA GATGTTTCTGCGCCCTTCTC
The Mut probe (primer 54171) anneals over the nucleotide sequence containing mouse genomic variations rs27362105.
Wt Sequence (with deletion in brackets):
GTGGAAAGGGAATTAAATTTCACATATACATGTATTTAAAATCATGCATATATTTAGTATAAGTGTCCCCAAAT[attgtcaggaaaggattggccattttggctgtatgatcagcacccaacagagaatattaaaataggagatttaataaatattgttggaaagaattaaatgaatgaaaaactgagggcatcaatcagttatttccaaaatattgcgatacgctattgatacttttcttttttgtttttgtttgcttgtttgattcttttcttaacttgactcccccccccccccccattgttcccaggtagcttagccaacatggctgcctccttgccgtctaccctgagcttcagttctgcacccgatgaaattcaac……..cggggcctgggcaagtgagggaaatgaatcgggtaacaagctgtttagacggtttcggaaaatgaatgccaggcagtccaagtgctatgagatggaagatgtcctgaaacatcactggctgtatacttcaaaatacctccagaagtttatgaatgctcataacgcgttaaaaagctctgggtttaccatgaactcaaaggagaccttaggggaccctttgggcattgaggactctctggaaagccaagattcaatggagttttaaataggatctccacatagaagttggtatttgccaatgtgttttcctttgggttgcagtgaggtcttctcctagcacctagcacattgccatgtgggtgggtcttatcacccaaggggtgacatgttggagtgagaggccagaggtgctgtcacct]GGTCAGGGATCGATAGTTGACAAGATGAGATTGCCTGAGCCGTTTAGTGAGGCCAGAAGAGCAACAGGAGAAATCAGTT
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
53511 | AAA GAG ACT TTG GTG CAT ATC AA | Wild type Forward | A | |||
53512 | GAT GTT TCT GCG CCC TTC TC | Wild type Reverse | A | |||
53513 | Fluorophore-1 | CTC GCC AGC ATC TCC TGT C | Quencher-1 | WT Probe | ||
53514 | AAA GGG AAT TAA ATT TCA CAT ATA CA | Mutant Forward | A | |||
53515 | CAG GCA ATC TCA TCT TGT CA | Mutant Reverse | A | |||
54171 | Fluorophore-2 | CCC AAA TGG TCA GGG ATC GA | Quencher-2 | MUT Probe |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
53511 | 0.40 uM |
53512 | 0.40 uM |
53514 | 0.40 uM |
53515 | 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 |